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REACH

Salicylic acid

EC number: 200-712-3 | CAS number: 69-72-7

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The assessment of salicylic acid is based on read-across data from studies on MeS and ASA. The studies used in the assessment are summarised below:

3-generation study (Collins et al., 1971), MeS, male and female Osborne-Mendel rats, 500, 1500, 3000 and 5000 ppm (equivalent to 22.5, 67.5, 135, 225 mg/kg bw/d as salicylic acid) in the diet: No statistically significant decrease in fertility index was reported at any dose for any generation.
2-generation study (Abbott & Harrisson, 1978), MeS, male and female Wistar rats, 2500 and 5000 ppm (equivalent to 113 and 225 mg/kg bw/d as salicylic acid) in the diet: Non-significant decrease in mating performance for the first generation.
2-generation study (Abbott & Harrisson, 1978), MeS, male and female mice, 2500 and 5000 ppm (equivalent to 324 and 648 mg/kg bw/d as salicylic acid) in the diet: No adverse effects were reported on any reproductive parameter.
2-generation study,( NTP, 1984a) continuous breeding protocol , MeS, CD-1 mice, 25, 50 and 100 mg/kg bw/d (22.5, 45 and 90 mg/kg bw/d as salicylic acid) by gavage: No effects on fertility were reported.
1-generation study (NTP, 1984b), continuous breeding protocol , MeS, CD-1 mice, 100, 250 and 500 mg/kg bw/d (90, 225 and 450 mg/kg bw/d as salicylic acid): No effect on fertility index.
Fertility test, (Schardein et al., 1969), ASA , male and female rats, A single dose level of 0.4% in the diet (210 mg/kg bw ASA, equivalent to 161 mg/kg bw as salicylic acid): ASA did not significantly affect male or female fertility. This dose caused moderate bw depression in males and severe bw depression in females.

The studies showed a number of deficiencies in relation to current TGs in terms of parameters studied, but the results were consistent. No statistically significant effect on fertility was reported in any study. In addition, 2-year chronic toxicity studies in rats and dogs (Webb, 1963) showed no abnormalities in sexual organs (testes/prostate or ovaries/uterus).

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data are available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study is similar to OECD 416, several currently recommended parameters were not assessed, but the study 2 years/oral/rat (Webb and Hansen, 1963 (reliability: 2) was used to supplement some observations.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

several deficiencies in relation to OECD Guideline 416 in terms of parameters studied

GLP compliance:

Limit test:

Species:

rat

Strain:

Osborne-Mendel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: (P) x wks; (F1) x wks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet: ad libitum (Purina Laboratory Chow)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): the diet was prepared every 14 days in a manner identical to that of Webb and Hansen (1963) and according to the results of Jones et al (1962).
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

VEHICLE: none

Details on mating procedure:

no data are available

Analytical verification of doses or concentrations:

Details on analytical verification of doses or concentrations:

none

Duration of treatment / exposure:

100 days before the first mating and then throughout the experiment.

Frequency of treatment:

once/day

Details on study schedule:

no data are available.

Dose / conc.:

500 ppm

Remarks:

equivalent to 25 mg/kg bw as MeS, or 22.5 mg/kg bw as SA; Basis: nominal in diet

Dose / conc.:

1 500 ppm

Remarks:

equivalent to 75 mg/kg bw as MeS, or 67.5 mg/kg bw as SA; Basis: nominal in diet

Dose / conc.:

3 000 ppm

Remarks:

equivalent to 150 mg/kg bw as MeS, or 135 mg/kg bw as SA; Basis: nominal in diet

Dose / conc.:

5 000 ppm

Remarks:

equivalent to 250 mg/kg bw as MeS, or 225 mg/kg bw as SA; Basis: nominal in diet

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

no data are available

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: No

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter ; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number of pups, stillbirths, live births, presence of gross anomalies,

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

not performed

Postmortem examinations (offspring):

SACRIFICE: no data

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations for the third generation only.

HISTOPATHOLOGY :
microscopic examination of livers and kidneys was performed.

Statistics:

the Chi-2 test was used to determine significant differences between each dose and the control for each mating in each generation.

Reproductive indices:

the fertility index (number of litters cast/number of females exposed to mating).

Offspring viability indices:

the viability index (number of liveborn/total number born)
the survival index (number alive at day 4/ number born alive)
the weaning index (adjusted number of day 21 survivors/number alive at day 4)

Clinical signs:

no effects observed

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS:
No clinical signs of toxicity were reported.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):

- Fertility index: no significant differences for any dose/1st generation. Appreciable decreases seen in 2nd and 3rd generations/5000 ppm.
- Average litter size/female: significant decreases were seen in the second generation in the second mating at 3000 ppm and in both mating at 5000 ppm. Although decreases were seen at 1500 ppm, they were not statistically significant because of the large variation in progeny between females within a group.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effect

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effect

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)

- The average number of liveborn young per female exposed to mating: Statistically significant differences were observed in both matings of the
second generation at 3000 ppm and at 5000 ppm.

- Viability index: "possible loss of young through stillbirths" in 2 matings/5000 ppm.

- Average no.surviving progeny/female, day 4: significant decreases occurred in both matings of the second generation at 3000 ppm and 5000 ppm

- Survival index, day 4: an adverse effect was observed in the second generation at the 3000 and 5000 ppm and in the first mating of the third generation at the same dose levels.

- Average no. progeny weaned/female, day 21: significant decreases were observed in the second generation at 3000 ppm in the first mating and at 5000 ppm in the first and second matings.

- Weaning index: "appreciable decrease" in 2nd generation/2nd litter/5000 ppm.

BODY WEIGHT:

- Average weanling weight,( day 21/sex): decreases in weight appeared consistently at the 3000 and 5000 ppm levels(in all generations).
GROSS PATHOLOGY (OFFSPRING)
no grossly visible abnormalities.

HISTOPATHOLOGY (OFFSPRING)
Histopathological examinations of the livers and kidneys of 3rd weanlings at 0, 3000 and 5000 ppm dose levels showed no indication of toxic effects

Dose descriptor:

NOAEL

Generation:

Effect level:

75 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

viability

mortality

Dose descriptor:

LOAEL

Generation:

Effect level:

150 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

viability

mortality

Reproductive effects observed:

not specified

Supplemental study: the results obtained after addition of calcium carbonate to methyl salicylate did not differ from those obtained after administration of methyl salicylate alone.

table 1 : fertility indexes of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0		500		1500		3000		5000
generation	mating	FI (a)	% (b)	FI	%	FI	%	FI	%	FI	%
1	1	20/20	100	20/20	100	20/20	100	20/20	100	20/20	100
	2	19/19	100	20/20	100	18/19	95	19/19	100	20/20	100
2	1	20/20	100	19/20	95	20/20	100	19/20	95	17/20	85
	2	19/19	100	19/20	95	19/19	100	19/20	95	10/13	77
3	1	20/20	100	18/20	90	18/19	95	19/20	95	17/19	89
	2	18/20	90	16/18	89	17/19	89	15/17	88	16/19	84

(a) Fertility Index (nb of litters cast/ nb of females exposed to mating)

(b) Percent females pregnant

table 2: average litter size of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0		500		1500		3000		5000
generation	mating	No. (a)	Av. (b)	No.	Av.	No.	Av.	No.	Av.	No.	Av.
1	1	208/20	10.4	211/19	11.1	207/20	10.4	235/20	11.8	188/18	10.4
	2	213/19	11.2	232/20	11.6	228/19	12.0	238/19	12.5	198/19	10.4
2	1	216/20	10.8	205/20	10.2	206/20	10.3	169/20	8.4	124/20	6.2 (c)
	2	226/19	11.9	204/20	10.2	189/18	10.5	187/20	9.4 (d)	86/13	6.6 (c)
3	1	192/20	9.6	188/19	9.9	172/19	9.1	170/20	8.5	179/19	9.4
	2	197/20	9.8	191/18	10.6	163/19	8.6	132/17	7.38	172/19	9.1

(a) Total number progeny/number females exposed to mating

(b) Average litter size per female exposed to mating

(d) significant at P<0.05

table 3: viability data for rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)	No.	Av.	VI (c, d)
1	1	208/20	10.4	1,00	211/19	11.1	1,00	195/20	9,8	0,94	229/20	11,4	0,97	167/18	9,3	0,88
1	2	213/19	11.2	1,00	231/20	11.6	1,00	226/19	11,9	0,99	237/19	12,5	1,00	189/19	9,9	0,95
2	1	215/20	10.8	1,00	203/20	10.2	0,99	203/20	10,2	0,99	164/20	8,2 (e)	0,97	106/19	5,6 (f)	0,85
2	2	225/19	11.8	1,00	203/20	10.2	1,00	189/18	10,5	1,00	182/20	9,1 (e)	0,97	82/13	6,3 (f)	0,95
3	1	188/20	9,4	0,98	184/19	9,7	0,98	160/19	8,4	0,93	164/20	8,2	0,96	174/19	9,2	0,97
3	2	196/20	9.8	1,00	186/18	10,3	0,97	155/19	8,2	0,95	118/17	6,9	0,89	166/19	8,7	0,97

(a) Total number liveborn/number females exposed to mating

(b) Average number liveborn per female exposed to mating

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

table 4: survival data of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	SI (c, d)	No.	Av.	SI	No.	Av.	SI	No.	Av.	SI	No.	Av.	SI
1	1	157/17	9,2	0,90	116/14	8,3	0,82	172/19	9,1	0,96	152/15	10,1	0,92	129/15	8,6	0,94
1	2	202/19	10,6	0,95	196/20	9,8	0,85	205/19	10,8	0,91	218/19	11,5	0,92	168/19	8,8	0,89
2	1	188/20	9,4	0,87	179/20	9	0,88	190/20	9,5	0,94	123/20	6,2 (e)	0,75	82/19	4,3 (f)	0,77
2	2	211/19	11,1	0,94	188/20	9,4	0,93	186/18	10,3	0,98	165/20	8,2 (e)	0,91	61/13	4,7 (f)	0,74
3	1	174/20	8,7	0,93	177/19	9,3	0,96	147/19	7,7	0,92	139/20	7	0,85	147/19	7,7	0,84
3	2	174/20	8,7	0,89	179/18	9,9	0,96	150/19	7,9	0,97	113/17	6,6	0,96	153/19	8,1	0,92

(a) Total number day 4 survivors / no. females exposed to mating

(b) Average number day 4 survivors per female exposed to mating

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

table 5: weaning data of rats fed methyl salicylate for 3 generations

dietary level (ppm)
		0			500			1500			3000			5000
gen.	mating	No. (a)	Av. (b)	WI (c, d)	No.	Av.	WI	No.	Av.	WI	No.	Av.	WI	No.	Av.	WI
1	1	154/17	9,1	0,98	114/14	8,1	0,98	172/19	9,1	1,00	151/15	10,1	0,99	129/15	8,6	1,00
1	2	183/19	9,6	0,91	187/20	9,4	0,95	203/19	10,7	0,99	191/19	10,1	0,88	164/19	8,6	0,98
2	1	176/20	8,8	0,94	168/20	8,4	0,94	188/20	9,4	0,99	121/20	6 (e)	0,98	74/19	3,9 (f)	0,90
2	2	200/19	10,5	0,95	173/20	8,6	0,92	179/18	9,9	0,96	160/20	8,0	0,97	48/13	3,7 (f)	0,79
3	1	170/20	8,5	0,98	172/19	9,1	0,97	146/19	7,7	0,99	122/20	6,1	0,88	137/19	7,2	0,93
3	2	170/20	8,5	0,97	179/18	9,9	1,00	149/19	7,8	0,99	111/17	6,5	0,98	144/19	7,6	0,94

(a) Total of no. of adjusted day 21 survivors/ no. females exposed. adjusted day 21 survivors = (no. alive at day 21)/ (no. kept at day 4) x no. alive at day 4

(b) Average number adjusted day 21 survivors per female exposed to mating

(d) Not analyzed for statistical significance

(e) significant at P<0.05

(f) significant at P<0.01

Conclusions:

Under the test conditions, MeS did not significantly reduce male or female fertility. MeS induced developmental toxicity, adverse effects on offspring viability was observed but with no evidence of increased incidence of malformations at the doses tested.The NOAELs were identified:
NOAEL (parental): 250 mg/kg bw/day
NOAEL (reproduction): 250 mg/kg bw/day
LOAEL (development): 150 mg/kg bw/day
NOAEL (development): 75 mg/kg bw/day

Executive summary:

In a 3 -generation study (Collins et al., 1971), rats were fed methyl salicylate at doses of 500, 1500, 3000 or 5000 ppm in the diet (equivalent to 25, 75, 150 or 250 mg/kg body weight as MeS, or 22.5, 67.5, 135, 225 mg/kg bw as SA) 100 days before the first mating and then throughout the experiment. No clinical signs of toxicity were reported at any dose level. No statistically significant decrease was reported in fertility index at any dose, however it was considered that there were "appreciable" decreases at 250 mg/kg in F2 and F3. Significant decreases were reported in average litter size, average number of live-born progeny, average numbers of survivors to PND4 and average number of weaning in the 150 and 250 mg/kg bw/day in F2. No external malformations were reported in pups of any litter and necropsy of the third generation weanlings showed no significant findings. The effects in the calcium carbonate supplement groups did not differ significantly from those of the groups fed MeS alone.

NOAEL (parental): 250 mg/kg bw/day

NOAEL (reproduction): 250 mg/kg bw/day

LOAEL (development): 150 mg/kg bw/day

NOAEL (development): 75 mg/kg bw/day

This study is similar to OECD guideline 416. Several currently recommended observations and parameters determinations were not performed, adult body weight and food consumption were not measured in this study, but were stated to have been unaffected at 5000 ppm in a previous study (Webb and Hansen, 1963), there are no data concerning possible effects in sex organs, corpora lutea, pre-implantation or post-implantation losses for any mating. However, no abnormalities in testes/prostate or ovaries/uterus were found in a 2 years study in rats (Webb and Hansen, 1963). Oestrous cycle data and sperm morphology/function data were not measured. Notwithstanding these deficiencies, the study is acceptable (reliability: 2) for reproductive risk assessment.

Reference 2

Endpoint:: fertility, other
Remarks:: based on test type
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: data not availalbe
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Study well documented and scientifically valid. No GLP. Only a single dose level used, causing significant toxicity in females. No data on the purity of acetylsalicylic acid
Reason / purpose for cross-reference:: reference to same study
Qualifier:: no guideline followed
Principles of method if other than guideline:: To determine the effect on male fertility, groups of 20 male rats were given acetylsalicylic acid (ASA) in the diet for 2 months (63 days), and then were exposed in a 1:1 ratio (overnight) to untreated females until at least 10 inseminated females were obtained. The dams were sacrificed on day 21. All pups were dissected for determination of external and internal gross abnormalities. To assess the effect on female fertility, groups of 30 females rats were given ASA in the diet for 14 days prior to mating and through weaning. They were exposed to untreated males (overnight) in a 1:1 ratio until at least 20 animals were inseminated. These dams were allowed to bear and wean a single litter.
GLP compliance:: no
Limit test:: yes
Species:: rat
Strain:: other: Holtzman
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: data not availalbe
- Age at study initiation: data not availalbe
- Weight at study initiation: data not availalbe
- Fasting period before study: data not availalbe
- Housing: data not availalbe
- Diet : ad libitum, Breeder's Ration (Parke, Davis& Company, Deroit, Michigan)
- Water : ad libitum
- Acclimation period: data not available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/- 1°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod : 12 hrs dark / 12 hrs light

IN-LIFE DATES: no data
Route of administration:: oral: feed
Vehicle:: not specified
Details on exposure:: data not available
Details on mating procedure:: - M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): data not available
Analytical verification of doses or concentrations:: not specified
Duration of treatment / exposure:: Exposure period: prior to mating
Premating exposure period (males): treated: 63 days prior to mating
Premating exposure period (females): treated: 14 days prior to mating and through weaning
Duration of test: The dams, inseminated by treated male prior to mating, were sacrificed on day 21 of gestation (1 day prior to term).
The dams, treated prior to mating, were allowed to bear and wean a single litter.
Frequency of treatment:: daily
Details on study schedule:: Number of generation studies: 1
Dose / conc.:: 0.4 other: % in the diet
Remarks:: equivalent to 210 mg/kg for female and 209 mg/kg for male; Basis: nominal conc.
No. of animals per sex per dose:: at least 10 inseminated females (male fertility test)
at least 20 inseminated females (female fertility test)
Control animals:: yes, plain diet
Details on study design:: no data
Positive control:: No
Parental animals: Observations and examinations:: No data
Oestrous cyclicity (parental animals):: No data
Sperm parameters (parental animals):: No data
Litter observations:: STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (only with female fertility test)
- If yes, maximum of 8 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality

GROSS EXAMINATION OF DEAD PUPS:
no
Postmortem examinations (parental animals):: No data
Postmortem examinations (offspring):: SACRIFICE
No data

GROSS NECROPSY
No

HISTOPATHOLOGY / ORGAN WEIGTHS
No
Statistics:: No data
Reproductive indices:: data not available
Offspring viability indices:: data not available
Clinical signs:: not specified
Body weight and weight changes:: not specified
Food consumption and compound intake (if feeding study):: not specified
Organ weight findings including organ / body weight ratios:: not specified
Histopathological findings: non-neoplastic:: not specified
Other effects:: not specified
Reproductive function: oestrous cycle:: not specified
Reproductive function: sperm measures:: not specified
Reproductive performance:: not specified
Dose descriptor:: other: NOAEL parental males
Effect level:: < 210 mg/kg bw/day
Remarks on result:: other: Generation not specified (migrated information)
Dose descriptor:: other: NOAEL parental females
Effect level:: < 210 mg/kg bw/day
Remarks on result:: other: Generation not specified (migrated information)
Clinical signs:: not specified
Mortality / viability:: mortality observed, treatment-related
Body weight and weight changes:: effects observed, treatment-related
Sexual maturation:: not examined
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: not specified
Histopathological findings:: not specified
Dose descriptor:: NOEL
Generation:: F1
Effect level:: < 210 mg/kg bw/day
Reproductive effects observed:: not specified
Conclusions:: Under the conditions of this test, aspirin did not induce effect on male or female fertility.
Executive summary:: In a reproduction study (Schardein et al., 1969), aspirin were given to rats in order to study the effect on male and female fertility. To determine the effect on male fertility, groups of 20 male rats were given diet containing 0.4 % ASA (209 mg/kg) for 2 months (63 days), and then exposed in a 1:1 ratio (overnight) to untreated females until at least 10 inseminated females were obtained. On day 21 of pregnancy, dams were sacrificed and all pups were dissected. To assess the effect on female fertility, groups of 30 female rats were given diet containing 0.4 % ASA (210 mg/kg) for 14 days prior to mating and through weaning. They were exposed to untreated males (overnight) in a 1:1 ratio until at least 20 animals were inseminated. These dams were allowed to bear and wean a single litter. Results with fertility study in male rats show no significant effect in fertility rate among the animals of aspirin groups compared to the controls. There were no abnormalities in pups of any of the group. Results with fertility study in female rats show also no effect on fertility rate among aspirin group compared to control group. All pregnancies in females treated with aspirin terminated in resorption of all fetuses. Under the conditions of this test, aspirin did not induce effect on male or female fertility.

Reference 3

Endpoint:: two-generation reproductive toxicity
Remarks:: based on test type
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: no data are available
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: see 'Remark'
Remarks:: This study is similar to OECD 416, Several currently recommended observations and parameter determinations were not performed. Notwithstanding these deficiencies, the study is acceptable (reliability: 2) for reproductive risk assessment (fertility) associated with the 3-generation study (Collins and al, 1971) and the chronic study in rat conducted by Webb and Harrisson, 1963.
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:: yes
Remarks:: several deficiencies in relation to OECD Guideline 416 in terms of parameters studied
GLP compliance:: no
Limit test:: no
Species:: mouse
Strain:: not specified
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet: no data
- Water: no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): due to the volatility of MeS only enough diet for one week was prepared at any one time.
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

VEHICLE: none
Details on mating procedure:: no data are available.
Analytical verification of doses or concentrations:: no
Details on analytical verification of doses or concentrations:: none
Duration of treatment / exposure:: 30 days before the first mating and then throught the experiment.
Frequency of treatment:: daily
Details on study schedule:: - F1 parental animals not mated until 4 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 4 weeks.
- Age at mating of the mated animals in the study: 12 weeks
Dose / conc.:: 0.25 other: %
Remarks:: equivalent to 2500 ppm or 357 mg/kg bw MeS, or 324 mg/kg bw as SA; Basis: nominal in diet
Dose / conc.:: 0.5 other: %
Remarks:: equivalent to 5000 ppm or 714 mg/kg bw MeS, or 628 mg/kg bw as SA; Basis: nominal in diet
No. of animals per sex per dose:: 25/sex/dose (F0)
30/sex/dose (F1)
Control animals:: yes, concurrent no treatment
Details on study design:: no data are available
Positive control:: no data
Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS: no

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: no

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no
Oestrous cyclicity (parental animals):: not performed
Sperm parameters (parental animals):: not performed
Litter observations:: STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter, excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities,

GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):: SACRIFICE
- Male animals: no data
- Maternal animals: All surviving animals, after the last litter of each generation was weaned.

GROSS NECROPSY: not perfomed

HISTOPATHOLOGY / ORGAN WEIGHTS: not performed.
Postmortem examinations (offspring):: SACRIFICE
no data

GROSS NECROPSY
- Gross necropsy consisted of external examination.

HISTOPATHOLOGY / ORGAN WEIGTHS: no data
Statistics:: the analyses of variance, student-test and determination of P values.
Reproductive indices:: - Reproduction index: no. weaned 21 days/no. liveborn x 100.
Offspring viability indices:: 1- Stillborn index: no. Stillborn/total born x100
2- Viability: no. alive 5 days /no. liveborn x100
Clinical signs:: not examined
Body weight and weight changes:: not examined
Food consumption and compound intake (if feeding study):: not examined
Organ weight findings including organ / body weight ratios:: not examined
Histopathological findings: non-neoplastic:: not examined
Other effects:: not examined
Reproductive function: oestrous cycle:: not examined
Reproductive function: sperm measures:: not examined
Reproductive performance:: effects observed, treatment-related
Dose descriptor:: NOAEL
Effect level:: 250 mg/kg bw/day
Remarks on result:: other: Generation: reproduction (migrated information)
Dose descriptor:: NOAEL
Effect level:: 250 mg/kg bw/day
Remarks on result:: other: Generation: development (migrated information)
Clinical signs:: not examined
Mortality / viability:: mortality observed, treatment-related
Body weight and weight changes:: not examined
Sexual maturation:: not examined
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: effects observed, treatment-related
Histopathological findings:: not examined
Reproductive effects observed:: not specified
Conclusions:: Under the test condition, no adverse reproduction effects were noted in mice at dietary levels of 125 and 250 mg/kg bw/day MeS.The NOAEL of 250 mg/kg bw/day was identified.
Executive summary:: A two-generation study similar to OECD guideline 416 was conducted by Abbott and Harrisson (1978), on methylsalicylate using 25 F0 mice/sex/dose and 30 F1 mice/sex/dose. The F0 mice were fed MeS at dietary concentrations of 0.25 or 5% (equivalent to 125 or 250 mg/kg body weight) from 30 days before the first mating and throughout the entire study period. F0 and F1 mice were each mated twice. Reproduction parameters (total born, live born, live at 5 days and at weaning) and pup abnormalities were monitored. No effects on any parameter were reported at either dose level. Therefore, the NOAEL (all endpoints) was 250 mg/kg bw/day, the highest dose tested in the study.

Reference 4

Endpoint:: two-generation reproductive toxicity
Remarks:: based on test type
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: no data are available
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: see 'Remark'
Remarks:: This study is similar to OECD 416, Several currently recommended observations and parameter determinations were not performed. Notwithstanding these deficiencies, the study is acceptable (reliability: 2) for reproductive risk assessment (fertility) associated with the 3-generation study (Collins and al, 1971) and the chronic study in rat conducted by Webb and Hansen, 1963.
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:: yes
Remarks:: several deficiencies in relation to OECD Guideline 416 in terms of parameters studied
GLP compliance:: no
Limit test:: no
Species:: rat
Strain:: Wistar
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Manor Farm
- Age at study initiation: for F0: 60 days old
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually
- Diet: ad libitum (Purina Diet)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: no data
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): due to the volatility of MeS only enough diet for one week was prepared at any one time.
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

VEHICLE: none
Details on mating procedure:: - M/F ratio per cage: mating was accomplished by placing a male with a femal of the same test group.
- Length of cohabitation: 1 week
Analytical verification of doses or concentrations:: no
Details on analytical verification of doses or concentrations:: none
Duration of treatment / exposure:: 60 days before the first mating and then throughout the experiment
Frequency of treatment:: daily.
Details on study schedule:: - F1 parental animals not mated until 4 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 4 weeks.
- Age at mating of the mated animals in the study: 12 weeks
Dose / conc.:: 0.25 other: %
Remarks:: equivalent to 2500 ppm or 125 mg/kg bw MeS, or 113, 225 mg/kg bw as SA; Basis: nominal in diet
Dose / conc.:: 0.5 other: %
Remarks:: equivalent to 5000 ppm or 250 mg/kg bw MeS, or 113, 225 mg/kg bw as SA; Basis: nominal in diet
No. of animals per sex per dose:: 25/sex/dose (F0)
30/sex/dose (F1)
Control animals:: yes, concurrent no treatment
Details on study design:: no data are available
Positive control:: no data
Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS: no

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: no

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no
Oestrous cyclicity (parental animals):: not examined
Sperm parameters (parental animals):: not examined
Litter observations:: STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter, excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities,

GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):: SACRIFICE
- Male animals: no data
- Maternal animals: All surviving animals, after the last litter of each generation was weaned.

GROSS NECROPSY: not perfomed

HISTOPATHOLOGY / ORGAN WEIGHTS: not performed.
Postmortem examinations (offspring):: SACRIFICE
no data

GROSS NECROPSY
- Gross necropsy consisted of external examination.

HISTOPATHOLOGY / ORGAN WEIGTHS: no data
Statistics:: the analyses of variance, t-test and determination of P values.
Reproductive indices:: - Reproduction index: no. weaned 21 days/no. liveborn x 100.
Offspring viability indices:: 1- Stillborn index: no. Stillborn/total born x100
2- Viability: no. alive 5 days /no. liverborn x100
Clinical signs:: not examined
Body weight and weight changes:: not examined
Food consumption and compound intake (if feeding study):: not examined
Organ weight findings including organ / body weight ratios:: not examined
Histopathological findings: non-neoplastic:: not examined
Other effects:: not examined
Reproductive function: oestrous cycle:: not examined
Reproductive function: sperm measures:: not examined
Reproductive performance:: effects observed, treatment-related
Clinical signs:: not examined
Mortality / viability:: mortality observed, treatment-related
Body weight and weight changes:: not examined
Sexual maturation:: not examined
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: effects observed, treatment-related
Histopathological findings:: not examined
Reproductive effects observed:: not specified
Conclusions:: Under the test conditions, MeS did not significantly modify fertility rate, NOAEL (reproduction) and NOAEL (parental) was 250 mg/kg bw/day, but
induced developmental toxicity: adverse effects on offspring viability was observed but with no evidence of increased incidence of malformations at the doses tested.
Executive summary:: A two-generation study similar to OECD guideline 416 was conducted by Abbott and Harrisson (1978), on methylsalicylate using 25 F0 Wistar rats/sex/dose and 30 F1 rats/sex/dose. The F0 rats were fed MeS at dietary concentrations of 0.25 or 5% (equivalent to 125 or 250 mg/kg body weight as MeS; 113 or 225 mg/kg bw as SA) from 60 days before the first mating and throughout the entire study period. F0 and F1 rats were each mated twice. No gross abnormalities were observed for neonates of any litter, and all surviving to weaning were normal in respect to growth, appearance, and behavior. At the dose of 125 mg/kg bw, the only reported effect of MeS treatment was a decrease in litter size. At 250 mg/kg bw/day, decreases in mating performance, reproductive and viability indices were noted but these findings were not statistically significant, and deaths between birth and postnatal day 5 were increased. In this study, The NOAEL (parental) and the NOAEL (reproduction) was 250 mg/kg bw/day, but the NOAEL (development) level could not be determined, given the results observed at lower dose, (a decrease in litter size).

Reference 5

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 1982/03/17 to 1982/12/21.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In the present study, the reproductive toxicity of MeS was evaluated according to the NTP "Fertility Assessment by Continuous Breeding (FACB)" protocol. GLP study, the purity is indicated. The study is acceptable for the MeS fertility assessment.

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this 2-generation study, MeS was administered in CD-1 Mice by gavage according to the NTP continuous breeding protocol at dose levels of 25, 50 and 100 mg/kg bw/day MeS (22.5, 45 and 90 mg/kg bw/day as SA) for 7 days prior to mating then for a 98 day cohabitation period and 21-day segregation periods. The FACB consists of four related tasks, not all of which are necessarily performed for a given compound. These tasks include, Task 1, dose finding; Task 2, continuous breeding phase, Task 3, identification of the affected sex and Task 4, offspring assessment.

GLP compliance:

yes

Limit test:

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston, NY).
- Age at study initiation: 11 weeks.
- Weight at study initiation: 35.24 +/- 0.72 g
- Fasting period before study: no data
- Housing: for the first week, animals will be housed five per cage by sex. subsequently, the females and males from the same dose group will be randomly paired and cohabited for 100 days., one breeding pair per cage (i.e., 20 breeding pairs per dose group). Thereafter each male and female will be housed individually for a period of 20 days.
- Diet: ad libitum (purina certified pelleted Rodent chow)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (C°): 22
- Humidity (%): no data (relative humidity will be monitored on 31-day recording hygrothermographs (Model H311, Weathermeasure Corp, Sacramento, CA located in the animal holding rooms for this study)
- Air changes (per hr): 12 to 14 times per hour.
- Photoperiod (hrs dark / hrs light): lights in each animal room were on 0700 to 2100 hours.

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): due to the volatility of MeS only enough diet for one week was prepared at any one time.
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: Each formulated sample will be refrigerated prior to and between dosing. An aliquot will be removed for dosing and warmed to room temperature.

VEHICLE: corn oil
- Amount of vehicle (if gavage): 30 mg/ml
- Lot/batch no. (if required): no data
- Purity: no data
- Source: Mazola.

Details on mating procedure:

- M/F ratio per cage:
1-Task2: one breeding pair will be housed per cage: 40 breeding pairs in the control group, and 20 breeding pairs per group for the three groups receiving test chemical.
2-Task4: a male and female/cage from different litters within treatment groups
- Length of cohabitation:
1- Task2: 100 days
2-Task4: from 1 to 7 days, depending upon when a copulatory plug was detected.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how):
1- Task2: after 98 days of cohabitation, each pregnant female was isolated, housed individullay for 21 days.
2- Task4: after 1 to 7 of cohabitation, the pairs were separated and the females were allowed to deliver their litters.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The MeS concentration will be determined by the following formula:
concentration (mg/ml) = dose level (mg/kg)/dose volume (10.0 ml/kg)
Midwest Research Institute performed the analysis of the dosage formulations, all data are present in the NTP report.

Duration of treatment / exposure:

For 7 days prior to mating then for a 98 day cohabitation period and 21-day segregation periods.

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until 4 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 3 weeks.
- Age at mating of the mated animals in the study: 11 weeks

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- 20/sex/dose for MeS groups
- 40/sex/dose for vehicle control group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: prior to commencing the reproductive study, a 14 day preliminary range-finding study was performed. The maximum tolerated dose (MTD) in male and female CD-1 mice was determined.

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: yes

BODY WEIGHT: yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no

Oestrous cyclicity (parental animals):

not performed

Sperm parameters (parental animals):

Parameters examined in F1 male parental generations:
testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
-The total number of pups/litter was adjusted by analysis of covariance.

PARAMETERS EXAMINED
The following parameters were examined in F1:
number and sex of pups, live births, stillbirths, weight gain

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All adult males were killed by asphixiation with CO2 and discarded at the conclusion of cohabitation in Task2 after the decision to perform Task 4 was made.
- Maternal animals: low dose females were killed by asphyxiation with CO2 and discarded at the conclusion of the 98-day cohabitation period. Mid dose adult females were killed and discarded on week 2 of Task 4. The high dose weanlings would survive until the time they were to be bred.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals (mid dose: 25 and 50 mg/kg Mes) and the F2 offspring were sacrificed at 127 +/- 8 days of age.

Statistics:

The Kruskal- Wallis analyses of variance, a Chi Square test, ANOVA test and Duncan's Multiple Range Test.

Reproductive indices:

- Fertility Index (%) = No. Fertile/ No. Cohabited X 100
- Mating Index (%) = No. with Copulatory plugs/ No. Cohabited X 100

Offspring viability indices:

No data are available.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Task 2:
- one parental male and female in the control group, one parental female in the 25 mg/kg Ms day/group, and three parental in the 100 mg/kg bw/day died prior to the conclusion of the continuous breeding phase. The random distribution of deaths across treatment groups suggested that they were not treatment-related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- in the F1 parental mice (Task4): the body weight for males and females were unaffected by MeS treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
-In the F1 parental mice: Sperm assessment indicated no significant difference in the % motile sperm, sperm concentration or % abnormal sperm in the cauda epididymis between male mice exposed to 0 or 100 mg/kg bw/day MeS.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
-The Fertility indices were comparable to than those of the controls. One control female died between 15 and 18 weeks of study and is not included in the 18 week data. (Task2)
- The percentage of pairs with the female having a copulatory plug was 76% in the high dose pairs compared to 95% in the control pairs. The difference was not statistically significant.
- In the pairs cohabited, 17 out of 19 control females were fertile compared to 11 out of 17 high dose females. This difference was marginally significant (p = 0.083)

ORGAN WEIGHTS (PARENTAL ANIMALS):
- There were no significant differences in liver and pituitary weights between the control and 100 mg/kg MeS treatment in male and female mice.
- The brain weight was increased and reproductive tract weight was decreased in female mice given 100 mg/kg bw/day MeS. The difference between the two groups appeared to be due to biological variation, it was not considered to be of toxicological significance.

GROSS PATHOLOGY and HISTOPATHOLOGY (PARENTAL ANIMALS):
- No-treatment-related gross or histopathologic lesions were noted for the pituitary, testis, epididymis, prostate or seminal vesicles in male mice, or for the pituitary, ovary, oviduct, uterus or vagina in female mice.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Remarks on result:

other: Generation: reproductive effects (migrated information)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- No significant differences existed between the groups with respect to average number of litters per breeding pair (Task2)
- Litter size was comparable to that of controls (Task 2 and Task4).
- The proportion of pups born alive per litter was not found to differ significantly among the dose groups (Task2, Task4)
- No effect on number of male pups (Task2). The high dose pairs produced a smaller number of males in the pups born alive (47% vs 55%), but the difference was not statistically significant (Task4).
- Adjusted Mean pup body weights:
Task 2:
- at 15 weeks, mean female pup weight in the high dose group was marginally increased relative to the control
- The increased female pup weight in the high dose group was not observed at 18 weeks.
Task4:
- Average pup weights in the high dose group did not differ significantly from controls.

- Unadjusted Mean pup weights:
Task2: there were no significant differences in mean pup weight.
Task4: Average pup weights in the high dose group was comparable to the controls

Dose descriptor:

NOAEL

Generation:

Effect level:

100 mg/kg bw/day

Reproductive effects observed:

not specified

Table 1: Fertility of pairs during continuos Breeding (Task 2)- Premating (1 week) and cohabitation (14 weeks) periods

Treatment Group (mg/kg/day)	No. Fertile/No. Cohabited	Fertility Index (%)
0	38/39 (b)	97
25	19/19 ( c)	100
50	16/16 (d)	100
100	17/17 ( e)	100

b: one male and female from different pairs died prior to the conclusion of the cohabitation period during Task 2.

c: one female died prior to the conclusion of the cohabitation period during Task 2

d: two females and three males died prior to the conclusion of the premating or cohabitation period during Task2

e: Three males died prior to the conclusion of the cohabitation period during Task2

Table 2: Mating and Fertility of pairs of task 2 offspring: effect of continuous exposure to MeS (task 4)

Treatment Group (mg/kg/day)	No. With copulatory plugs/No. cohabited	Mating Index (%)	No. Fertile/No, Cohabited	Fertility Index (%)
0	18/19 ( c)	95	17/19 ( c)	89
100	13/17 (d)	76	11 /17 (d)	65

c: one female died prior to the conclusion of Task4
d: in the 100 mg/kg/day group only 17 males were available for mating in Task4

Table3: Sperm analysis for Task 2 Male Offspring: Effect of Continuous Exposure to MeS (Task4)

	Treatment Group (mg MeS/kg/day)
Parameter (Mean +/- S.E)	0	100
% Motile Sperm	90.70 +/- 1,90 (20) (b)	84 +/- 5,54 (17)
Sperm Concentration (No, sperm x 1000/mg caudal tissue	478,34 +/- 33,45 (20)	454,54 +/- 41,66 (17)
% Abnormal Sperm (c)	3,55 +/-0,44 (20)	2,99 +/- 0,64 (17)
% Tailless Sperm (d)	24,43 +/- 1,33 (20)	25,47 +/- 2,03 (17)

b: number of observations indicated in parenthesis

c: Tailless sperm not included in determination of % abnormal sperm

d: Tailless sperm were found to be an artifact of the method used to prepare sperm suspensions. Maceration of the cauda epididymis appeared to cause the loss of tails from many of the spermatozoa.

Conclusions:

Under the test condition, no effects on fertility, number of pups per litter, percentages of live pups or pup weight. Necropsy of F1 mice revealed no effects on body or organ weights or sperm motility, density or morphology. A NOAEL for reproductive effects of 100 mg/kg body weight/day and the NOAEL (development): 100 mg/kg bw/day were identified.

Executive summary:

A two-generation study according to the NTP continuous breeding protocol (Task1, Task2 and Task 4) was conducted on CD-1 Mice by gavage. In this study mice were administered MeS by gavage (in corn oil) at 25, 50 or 100 mg/kg/day during the 7 -day pre-mating and a 98 -day cohabitation period (Task2) (NTP, 1984a). Treatment was not associated with any adverse effects on fertility, number of pups/litter, percentage of live pups, or on pup weight. Necropsy of the F1 animals, reared and dosed with MeS, revealed no adverse effects on terminal body and organ weights or on sperm motility, density and morphology. A NOAEL for reproductive effects of 100 mg/kg body weight/day was identified.

- Task 1 (14-day range-finder)

- Task 2 (1-generation study)

- Task 4 (2nd -generation)

Reference 6

Endpoint:

toxicity to reproduction

Remarks:

other: one generation + fertility

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 1984/03/16 to 1982/12/23.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

In the present study, the reproductive toxicity of MeS was evaluated according to the NTP "Fertility Assessment by Continuous Breeding (FACB)" protocol. GLP study, the purity is indicated. Several deviations in protocol are mentioned, but the study is acceptable in accordance with the NTP 1984a study for the MeS fertility assessment at different dose levels.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study, MeS was administered in CD-1 Mice by gavage according to the NTP continuous breeding protocol at dose levels of 100, 250 and 500 mg/kg bw/day MeS (90, 225 and 450 mg/kg bw/day as SA) for 7 days prior to mating then for 14 weeks of cohabitation and 3 weeks thereafter. The FACB consists of four related tasks, not all of which are necessarily performed for a given compound. These tasks include, Task 1, dose finding; Task 2, continuous breeding phase, Task 3, identification of the affected sex and Task 4, offspring assessment.

GLP compliance:

yes

Limit test:

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Inc. (Kingston, NY).
- Age at study initiation: eleven- week old.
- Weight at study initiation: 35.6 +/- 0.76 for males and 26.3 +/- 0.66 for females.
- Fasting period before study: no data
- Housing: 2 animals per cage.
- Diet: ad libitum (purina certified pelleted Rodent chow)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (C°): between 20-25 °C
- Humidity (%): between 20 to 70%
- Air changes (per hr): at least 10 or more air changes per hour of HEPA-filtered air.
- Photoperiod (hrs dark / hrs light): 10 hrs dark/14 hrs light.

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): every two weeks
- Mixing appropriate amounts with (Type of food): dosing solutions were formulated by mixing the test article directly into different proportions of corn oil.
- Storage temperature of food: Each formulated sample will be refrigerated prior to and between dosing. An aliquot will be removed for dosing and warmed to room temperature.

VEHICLE: corn oil
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): no data
- Purity: no data
- Source: Mazola.

Details on mating procedure:

- M/F ratio per cage:
1- For Task 2: one breeding pair will be housed per cage: 40 breeding pairs in the control group, and 20 breeding pairs per group for the three groups receiving test chemical.
2- For Task 3: one male mice, previously exposed to the MTD of the test article will be matched with control female mice, similarly one female mice previously treated with the MTD of the test article will be matched with control males. The remaining 20 control males will be randomly matched with the remaining 20 control females.
- Length of cohabitation:
1- For Task 2: 100 days
2- For Task 3: 7 days

- Proof of pregnancy: vaginal plugs.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how):
- Task 2: after 14 weeks of cohabitation, each pregnant female was isolated, housed individullay for 3 weeks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aliquots of various dosage formulations were sent to MRI for chemical analysis. reference aliquots were within 93 to 102 percent of the indicated MeS concentrations. These limits were considered acceptable. (The detailled reports describing analysis of various reference samples are present in Appendix III of NTP report)

Duration of treatment / exposure:

Task2: 7 days prior to mating then for 14 weeks of cohabitation period and 3 weeks thereafter.

Frequency of treatment:

daily

Details on study schedule:

not performed

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- 20/sex/dose for MeS groups
- 40/sex/dose for vehicle control group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: prior to commencing the reproductive study, a preliminary range-finding study (Task 1) was performed. The maximum tolerated dose (MTD) in male and female CD-1 mice was determined.

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS: no

BODY WEIGHT: yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no

Oestrous cyclicity (parental animals):

not performed

Sperm parameters (parental animals):

not performed.

Litter observations:

not performed.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after Task 3.
- Maternal animals: All surviving animals, after Task 3.

GROSS NECROPSY: no

HISTOPATHOLOGY / ORGAN WEIGHTS: not performed.

Postmortem examinations (offspring):

not performed.

Statistics:

the analyses of variance, student-test, determination of P values and non parametric test (Kruskal-Wellis).

Reproductive indices:

- Fertility Index (%): No. Fertile/No. Cohabite X 100.
- Mating Index (%): No. with Copulatory plugs/ No. Cohabited X 100.

Offspring viability indices:

not performed

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Task 2:
- Eleven animals died during Task 2, 3 in the control, 2 each in the 0.1 g/kg and 0.25 g/kg dose groups, and 4 in the 0.5 g/kg dose group. The cause of death varied from case to case but it was neither chemical nor dose related.
- No distinct treatment related symptoms of toxicity were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Task 2:
- MeS administered by gavage at 0.1, 0.25, and 0.5 g/kg dose levels had no apparent effect on male or female body weights.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Task 2:
The amounts of MeS and the volume of corn oil gavaged each day were once again based on the body weight at the beginning of each week.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Task 2:
- the fertility index in the control and various treatment groups varied between 95 to 100%, all breeding pairs except 1 in the 0.1 g/kg group delivered at least one litter.

- Task 3 (designed to identify the sex affected by the chemical exposure) was conducted twice: during the first trial the fertility index value for the control pairs was unusually low and, therefore, the experiment was repeated but it was still not possible to determinate which sex was affected by MeS treatment.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Remarks on result:

other: Generation: reproductive effect (migrated information)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
- There was a significant decrease (p<0.05) in the mean number of litters at the 0.5 g/kg MeS level.
- The average number of pups per litter, the proportion of pups born alive, and mean live pup weight values were also significantly reduced (p<0.05) in the 0.5 g/kg group compared to the corresponding controls.
- There was no significant effect on any of these parameters in the remaining two dose groups (only a 3% reduction in pup weight at 250 mg/kg).

Reproductive effects observed:

not specified

Table 1: Fertility of pairs during continuous breeding MeS (Task 2)

Treatment group	No, Fertile/ No, Cohabited	Fertility Index (%)

Control	38/38	100
0,1 g/kg	17/18	94
0,25 g/kg	18/18	100
0,50 g/kg	16/16	100

Conclusions:

Under the test condition, no effect on fertility index was observed. Adverse effects were observed in the litter analysis in the high dose groups, with only a 3% reduction in pup weight at the mid dose level. The NOAEL (reproductive effects): 100 mg/kg b.w/day and the NOAEL (parental): 500 mg/kg bw /day were identified.

Executive summary:

A study according to the NTP continuous breeding protocol (Task1, Task2, Task3) was conducted on CD-1 Mice by gavage. In this study mice were administered MeS by gavage (in corn oil) at 100, 250 or 500 mg/kg/day during the 7 -day pre-mating and a 14 weeks of cohabitation period and 3 weeks thereafter (Task 2), (NTP, 1984b). Exposure to MeS produced no adverse effect on fertility in Task 2. However, adverse effects were observed in the litter analysis in the mid and high dose groups. A significant decrease in average number of litters and a decrease in the proportion of pups born a live were seen in the high dose group. Dose related decreases were found in average litter size and average pup weight with the decreases being significant in the high dose group and marginally significant in the mid dose group. Fertility in the control group in the original Task 3 was so low (26%) that the animals were randomized again and another Task 3 was performed. In the second Task 3, only 41% of the controls were fertile. Due to the fertility problems in the control group and lack of significant results in the litter analyses, an affected sex cannot be determined. The NOAEL (reproductive effects): 100 mg/kg b.w/day was identified.

Task1:dose finding

Task2: continuous breeding phase

Task3: identification of the affected sex.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

No fertility studies are available on salicylic acid. Assessment of the potential of SA to impair fertility has therefore been based on read-across data from studies on Methyl salicylate (MeS) and Acetylsalicylic acid (ASA). The read-across approach is considered acceptable since the initial step in the metabolism of these salicylate compounds is hydrolysis to free salicylate (see section 7.1.1).

The potential for MeS to affect male and/or female fertility has been assessed in rats and mice in several multi-generation studies, with the study by Collins et al (1971) considered to be the key study for this endpoint.

In a 3-generation study (Collins et al., 1971), MeS was administered to male and female Osborne-Mendel rats in the diet at 500, 1500, 3000 and 5000 ppm (equivalent to 22.5, 67.5, 135, 225 mg/kg bw as SA). Parental generation rats were fed MeS for 100 days prior to mating, then throughout two mating, gestation and lactation periods. F1 and F1 rats received the test compound throughout the study, which terminated with the weaning of the F3 offspring. No statistically significant decrease was reported in fertility index at any dose for any generation. Adverse effects were reported on offspring, representing embryo-foetal toxicity primarily in terms of reduced viability (decreases in litter size, number of live-born progeny, number of survivors to PND4 and PND5 and number of survivors to weaning).

In a 2-generation study (Abbott & Harrisson, 1978), male and female Wistar rats received MeS in the diet at 2500 and 5000 ppm (equivalent to 113 and 225 mg/kg bw/day as SA) for 60 days prior to mating, then throughout the study. Each generation of rats was mated twice. This study reported a non-significant decrease in mating performance for the first generation, and reduced viability of pups.

Abbott and Harrisson also reported data on male and female mice exposed to MeS in the same manner at the same dietary concentration as rats (2500 and 5000 ppm (equivalent to 324 and 648 mg/kg bw/day as SA) from 30 days prior to mating. No adverse effects were reported on any reproductive parameter.

Two 2-generation studies have been conducted on MeS in CD-1 Mice by gavage according to the NTP continuous breeding protocol (NTP, 1984a, 1984b). At dose levels of 25, 50 and 100 mg/kg bw/day MeS (22.5, 45 and 90 mg/kg bw/day as SA) for 7 days prior to mating then for a 98 day cohabitation period, no effects were reported on fertility, number of pups per litter, percentages of live pups or pup weight. Necropsy of F₁ mice revealed no effects on body or organ weights or sperm motility, density or morphology. In a second study at the higher dose levels of 100, 250 and 500 mg/kg bw/day (90, 225 and 450 mg/kg bw/day as SA) there was no effect on fertility index. Reduced pup viability was reported at the high dose, with only a 3% reduction in pup weight at the mid dose level.

As conclusions on above studies, no statistically significant effect on fertility was reported in any study. Reduced embryo-foetal viability was reported at high maternally toxic dose levels, when parental toxicity refers to the systemic NOAELs.

The potential for effects on male and female fertility from ASA was reported by Schardein et al. (1969). This study used only a single dose level of 210 mg/kg ASA (161 mg/kg bw as SA) by oral gavage as positive control. Male rats were treated for 63 days prior to mating with untreated females. Female rats were treated for 14 days prior to mating with untreated males and up to weaning. ASA did not significantly affect male or female fertility at this dose which caused moderate bodyweight depression in males and severe bodyweight depression in females.

The studies above show a number of deficiencies in relation to current guidelines in terms of parameters studied, however their results are consistent in . In addition, 2-year chronic toxicity studies (Webb, 1963) in rats and dogs showed no abnormalities in testes/prostate or ovaries/uterus.

The adverse effects on reduced viability of offspring reported primarily in rats represent developmental toxicity rather than reduction of the fertility of either male or female animals. It can therefore be concluded that SA is not likely to have any significant adverse effect on fertility.

Discussion of human information

Human information generally doses not dissociate information on Fertility and on Developmental Toxicity. The effect of ASA on development has been studied in rats, mice and rabbits with results leading to the conclusion that there are considerable species differences in sensitivity, with the rat being the most sensitive species. Data on the effect of aspirin (ASA) in human pregnancy has been used to assess the relevance of the animal data for risk assessment. These data indicate that humans are less sensitive than rats to the effect of ASA and more comparable to rabbits in several points including ADME or protein binding. A rat key study (Gupta et al, 2003) administered aspirin (ASA) on GD6-17. Results from all studies showed that acetyl salicylic acid is embryotoxic at moderate maternal toxic doses and induces malformations at high maternally toxic doses. For effects in rabbits, the key study is Cappon et al (2003). There were no adverse effects on development at doses not causing severe maternal toxicity.

Well-designed epidemiological studies (Slone, 1976; Shapiro, 1976; Kozer, 2002) on the use of aspirin at up to the maximum recommended therapeutic dose of 4000 mg/day (equivalent to 66.7 mg/kg bw/day as ASA or 56 mg/kg/day as MeS) have largely demonstrated an absence of increased risk of adverse pregnancy outcome in terms of frequency of stillbirth, neonatal mortality, birth defects or developmental delay, despite widespread self-administration of aspirin during pregnancy. A meta-analysis of studies on the use of low-dose aspirin at 50-150 mg/day (Kozer, 2003) has demonstrated that this dose range is not associated with any adverse pregnancy outcomes, in terms of perinatal mortality, birth complications, congenital malformations or adverse effect on subsequent development. For pregnancies where there was moderate or high risk of pre-eclampsia and/or premature delivery, adverse pregnancy outcome rate was reduced with low-dose aspirin. There was no increased risk of early miscarriage with this dose regime. These data have been reviewed and completed by an Epidemiologist (Pr. D. BARD report to Novacyl, 2012) with a conclusion of no link between ASA use during pregnancy and reprotoxic effects. This absence of any clear evidence of adverse effects from aspirin on human development demonstrated in well-designed epidemiological studies despite widespread prescribed use and self-medication with aspirin at all stages of pregnancy over a period spanning several decades appears to indicate that humans are considerably less sensitive(ordifferent) than rats to the developmental toxicity of salicylate. Overall, it can be concluded that ASA does not adversely affect fertility and that the developmental toxicity reported in the rat is of very questionable (significance/ relevance) for humans. Since, ASA is not considered as reprotoxic for humans, no DNEL should be derived for this endpoint, the rabbit NOAEL being higher than the rat used.

Effects on developmental toxicity

Description of key information

In summary and according to the RAC opinion (2016) the results of the studies demonstrated that salicylic acid has an embryo-/foetotoxic effect in rats with dose-dependent growth delays, foetal death and malformations. The study in monkeys also showed teratogenic properties with ASA but with lower magnitude. By contrast, the effects in rabbits were limited to slight growth retardation and were present only at doses much higher than in the rats and monkeys. No skeletal malformations were reported and at the highest dose only one kit of a dam had hydrocephaly.

Overall, salicylic acid was shown to have teratogenic properties but with species differences in potency: strong in rats and lower in monkeys. In contrast, the teratogenic potential in rabbits was practically non-existent.

In human, no data are available with SA, but data on ASA exists. Acetylsalicylic acid has been used as a medicine for a very long time and consequently, it has been extensively studied. However, older as well as more recent studies have failed to arrive at a strong conclusion on the potential of ASA to induce malformations.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: no data are available
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: No GLP stated, purity unknown but this recent study is performed in an ICH guideline (CPMP/ICH/386/95) which requires compliance with GLP or equivalent standard.
Reason / purpose for cross-reference:: reference to other study
Qualifier:: according to guideline
Guideline:: other: ICH Topic S 5(R2)
Principles of method if other than guideline:: ASA was administered to pregnant New Zealand rabbits from gestation days GDs 7 to 19 (comparable to guideline 414) and as single high doses on GD 9, 10 or 11. Cesarean sections were performed on GD29, and the fetuses were examined for external, visceral, and skeletal development.
GLP compliance:: not specified
Limit test:: no
Species:: rabbit
Strain:: New Zealand White
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Covance Research Products, Inc (Denver, CO)
- Age at study initiation: 5 to 6.5 months
- Weight at study initiation: 2.8 kg
- Fasting period before study: 1 day
- Housing: rabbits were housed in stainless steel suspended wire cages.
- Diet: Purina Regular Rabbit Chow (Ralston, VA)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C ): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark /12 hrs light

IN-LIFE DATES: no data
Route of administration:: oral: gavage
Vehicle:: other: methylcellulose
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
no data

DIET PREPARATION
no data

VEHICLE
- Justification for use and choice of vehicle: no data
- Amount of vehicle (if gavage): 0.5%
- Purity: no data
- Source: Dow Chemical Co., Midland, MI
Analytical verification of doses or concentrations:: no
Details on analytical verification of doses or concentrations:: none
Details on mating procedure:: no data are available
Duration of treatment / exposure:: For the multiple study: from GD7 to GD19
For the single study: individual days, DG9, 10 or 11
Frequency of treatment:: Single daily doses
Duration of test:: around the time of gestation (29 days)
Dose / conc.:: 125 mg/kg bw/day
Remarks:: 96 mg/kg bw/day as salicylic acid (SA)
Dose / conc.:: 250 mg/kg bw/day
Remarks:: 192 mg/kg bw/day as SA
Dose / conc.:: 350 mg/kg bw/day
Remarks:: 268 mg/kg bw/day as SA
No. of animals per sex per dose:: For the multiple study: 20
For the single study: 10
Control animals:: yes, concurrent no treatment
Details on study design:: - Dose selection rationale:

For the multiple study: dose levels were based on findings from a dose range-finding study in pregnant rabbits. MTD = 350 mg/kg per day (3/20 animals died as result of treatment) for this dosing duration (13 days) in pregnant rabbits.

For the single study: ASA was administered to rabbits at higher dose > MTD = 350 mg/kg per day (during sensitive periods of cardiovascular development and midline closure)
Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were observed at least twice daily for morbidity and mortality during the study.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: it was determined on the day of arrival and then daily beginning on GD8.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: it was determined on the day of arrival and then daily beginning on GD8.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: the abdominal, thoracic and pelvic viscera , Uterus and ovaries
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:: - External examinations: Yes: all per litter
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No
Statistics:: several types of analyses were used for determining dose-response relations to ASA treatment:
Welch trend test (linear and in proportions) and analysis of covariance.
Indices:: no data are available
Historical control data:: no data are available
Details on maternal toxic effects:: Maternal toxic effects:yes

Details on maternal toxic effects:
In the repeated dose study, three does from the 350 mg/kg group died between GD14 and GD16. An additional doe in the 350 mg/kg group aborted on GD22.Necropsy examinations showed evidence of gastrointestinal toxicity(GI) for these does, consisting of dark red foci or pitted areas on the glandular mucosa of the stomach. Maternal body weight gain was significantly reduced in the mid and high dose groups from GD7 to GD13. Food consumption was significantly reduced throught the exposure duration in these groups.
In the single dose study, one doe from the 500 mg/kg group and one from the group 1000 mg/kg group that were dosed on GD10 and two does from the 750 mg/kg group dosed on GD 11 showed signs of GI toxicity. In this study, maternal toxicity was exhibited at all doses by reductions in body weight gain and food consumption for 3 days after treatment.
Dose descriptor:: NOAEL
Effect level:: 125 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
Dose descriptor:: NOAEL
Effect level:: 250 mg/kg bw/day
Basis for effect level:: other: developmental toxicity
Dose descriptor:: NOAEL
Effect level:: 350 mg/kg bw/day
Basis for effect level:: other: other:
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
In the repeated dose study, fetal body weight was significantly reduced at 350 mg/kg per day.
Fetal body weights were not affected by single doses of ASA on GD9, 10 or 11.
There were no treatment related external, visceral, or skeletal malformations associated with ASA administration throughout organogenesis (GD7 to 19) or single dose administered during critical developmental windows (even from treatment at up to 1000 mg/kg ).
Dose descriptor:: NOAEL
Effect level:: 350 mg/kg bw/day (nominal)
Sex:: male/female
Basis for effect level:: other: malformations
Dose descriptor:: NOAEL
Effect level:: 250 mg/kg bw/day (nominal)
Sex:: male/female
Basis for effect level:: fetal/pup body weight changes
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: Under the test conditions, ASA did not induce malformations in rabbits when it was administered as a single dose or during the period of organogenesis (GD7 to 19), even at doses causing significant maternal toxicity. The NOAELs were identified: NOAEL (maternal): 125 mg/kg bw/day NOAEL (malformations): 350 mg/kg bw/day NOAEL (development): 250 mg/kg bw/day.
Executive summary:: In an ICH-compliant study, Acetylsalicylic acid (ASA) was administered by oral gavage to pregnant New Zealand White rabbits at 125, 250 or 350 mg/kg bw/day (96, 192, 268 mg/kg as Salicylic acid (SA)), followed by termination on GD29 (Cappon and al, 2003). 3 does from the high and one doe from the mid dose died between GD14 and 16 and an additional doe aborted on GD22. Necropsy showed evidence of gastrointestinal toxicity. Maternal body weight gain was significantly reduced in the mid and high dose groups from GD7 to GD13. Food consumption was also reduced in these groups. There were no treatment-related effects on corpora lutea, implantation sites, pre-implantation losses or embryofoetal mortality. Mean foetal weight was significantly reduced in the high dose group. There were no treatment-related visceral or external anomalies. The same study also administered ASA at higher doses on individual days, DG9, 10 or 11.No treatment-related malformations were induced in rabbit fetuses by single dose administrations. The NOAELs were identified: NOAEL (maternal): 125 mg/kg bw/day NOAEL (malformations): 350 mg/kg bw/day NOAEL (development): 250 mg/kg bw/day.

Reference 2

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: no data are available
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: No GLP stated, purity unknown but this recent study is performed in an ICH guideline (CPMP/ICH/386/95) ) which requires compliance with GLP or equivalent standard.
Reason / purpose for cross-reference:: reference to other study
Qualifier:: according to guideline
Guideline:: other: ICH Topic S 5(R2)
Principles of method if other than guideline:: ASA was administered to pregnant Sprague Dawley rats from gestation days GDs 6 to 17 (comparable to guideline 414) and as single high doses on GD 9, 10 or 11. Cesarean sections were performed on GD21, and the fetuses were examined viscerally.
GLP compliance:: not specified
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Laboratory (Kingston, NY)
- Age at study initiation: no data
- Weight at study initiation: 225 to 250 g
- Fasting period before study: no data
- Housing: individually
- Diet : ad libitum (PMI 5002 meal)
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70 +/- 5°F (18°C)
- Humidity (%): 50 +/- 10 %
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hr light and dark cycle.

IN-LIFE DATES: no data
Route of administration:: oral: gavage
Vehicle:: other: 0.5% methyl cellulose
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

VEHICLE
- Justification for use and choice of vehicle: no data
- Amount of vehicle (if gavage): 0.5%
- Lot/batch no. (if required): no data
- Purity: no data
- Source: Dow Chemical Company, Midland, MI.
- CAS: R11596-0376
Analytical verification of doses or concentrations:: not specified
Details on analytical verification of doses or concentrations:: none
Details on mating procedure:: no data are available
Duration of treatment / exposure:: 1- for Single dose study: GD 9, 10 or 12
2- for multiple dose study: from day 6 to 17 of the gestation (period of organogenesis)
Frequency of treatment:: Single daily doses
Duration of test:: around 21 days
Dose / conc.:: 50 mg/kg bw/day
Remarks:: 38 mg/kg bw/day as salicylic acid (SA)
Dose / conc.:: 125 mg/kg bw/day
Remarks:: 96 mg/kg bw/day as SA
Dose / conc.:: 250 mg/kg bw/day
Remarks:: 192 mg/kg bw/day as SA
No. of animals per sex per dose:: - 7/female/dose for Single Dose Study
- 20/female/dose for Multiple Dose Study
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale:
1- for single dose study: study design and dose levels were based on the work of Kimmel et al. (1971).
2- for multiple dose study: a maternal range-finding study was conducted at doses of 50, 125, 250 or 500 mg/kg/day (n= 7/group). The MTD was considered to be 250 mg/kg. The doses of 50 and 125 mg/kg were chosen to provide a dose-response relationship.
Maternal examinations:: CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: uterus and ovaries
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: viable and dead fetuses were recorded for each dam.
Fetal examinations:: - External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: No
1- for single Dose study: Skeletal examinations were not perfomed in this study because the focus was to understand the potential of ASA to
produce cardiovascular defects and midline defects in rats.
2-for the multiple dose study: fetuses were retained for skeletal examination but not reported.
- Head examinations: Yes
Statistics:: - The dam was randomized to treatment
- Analyses of variables measured on individual pups were based on litter means. Data were analyzed using parametric and nonparametric trend tests, depending on the distribution characteristics of the variable under consideration.
Indices:: no data are available
Historical control data:: no
Details on maternal toxic effects:: Maternal toxic effects:yes

Details on maternal toxic effects:
In the multiple dose study, ASA produced a dose -dependent decrease in maternal body weight gain that was statistically significant in the mid (85% of control) and high (52% of control) dose groups. Food consumption was significantly reduced only at the high dose (85% of control).

In the single dose study, no maternal deaths were noted in any dose groups, there were no treatment related clinical signs or necropsy findings. Dose dependent decreases in body weight gain and food consumption were observed.
Dose descriptor:: NOAEL
Effect level:: 50 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
Dose descriptor:: NOAEL
Effect level:: 50 mg/kg bw/day
Basis for effect level:: other: developmental toxicity
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
In the multiple dose study:
the number of early resorptions and post-implantation loss was increased and foetal body weight was reduced in the high dose group (250 mg/kg bw/day). The malformations noted in fetuses from dams administered ASA on GDs 6 to 17 were statistically increased only in the high-dose group. These malformations included ablepharia , craniorachischisis, bent forepaw, kinked tail, protruding tongue, gastroschisis, ectopic adrenal, ventricular septal defect (VSD), diaphragmatic hernia, hypoplastic kidney and hypoplastic testis. No malformations were reported at 125 mg/kg.
In the single dose study:
Significant increases in malformations were reported only from 500 mg/kg for GD9 administration or from 625 mg/kg for GD10 or 11
Dose descriptor:: NOAEL
Effect level:: 50 mg/kg bw/day (nominal)
Sex:: male/female
Basis for effect level:: reduction in number of live offspring; skeletal malformations; visceral malformations
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: Under the test conditions, no significant strain differences in developmental anomalies between Wistar and SD rats were noted, with the exception of VSD in the SD rats and hydrocephalus in the Wistar rats. ASA was associated with increased incidences of malformations when administered at high dose, and with reduced offspring viability at lower dose during the period of organogenesis (GD6 to 17) in rat. The NOAEL (maternal): 50 mg/kg and the NOAEL (developmental): 50 mg/kg
Executive summary:: In an ICH-compliant study, ASA was administered by oral gavage to pregnant Sprague-Dawley rats at 50, 125 or 250 mg/kg bw/day(38, 96, 192 mg/kg as SA) on GD6 -17, followed by termination on GD21. Maternal bodyweight gain was significantly reduced in the mid and high dose groups. Food consumption was significantly reduced only at the high dose. The number of early resorptions and post-implantation loss was increased and foetal body weight was reduced in the high dose group. The number of viable fetuses was decreased in the mid and high dose groups. There was a significant increase in in the number of malformations only in the high dose. The same study also administered ASA at higher doses on individual days, DG9, 10 or 11. Significant increases in malformations were reported anly from 500 mg/kg for GD9 administration or from 625 mg/kg for GD10 or 11. The current study was conducted at doses similar to those selected by Kimmel et al. (1971), in part to compare the strain differences under a similar dosing regimen. There was generally concordance in major developmental anomalies between the two strains apart from hydrocephalus (only in Wistar starin) and ventricular septal defect (VSD) (only in SD strain). The NOAEL (maternal): 50 mg/kg and the NOAEL (developmental): 50 mg/kg

Reference 3

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: data not availalbe
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: No GLP. Purity of the studied products not stated. Study well described.
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Qualifier:: no guideline followed
Principles of method if other than guideline:: Teratogenic potential: To assess the teratogenic effect, animals were treated by gavage during the period of organogenesis (day 6 through day 18 of gestation).
GLP compliance:: not specified
Limit test:: no
Species:: rabbit
Strain:: Dutch
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: data not availalbe
- Age at study initiation: data not availalbe
- Weight at study initiation: data not availalbe
- Fasting period before study: data not availalbe
- Housing: data not availalbe
- Diet : ad libitum, Breeder's Ration (Parke, Davis& Company, Deroit, Michigan)
- Water : ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/- 1°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod : 12 hrs dark / 12 hrs light

IN-LIFE DATES: no data
Route of administration:: oral: gavage
Vehicle:: other: gum acacia
Details on exposure:: no data.
Analytical verification of doses or concentrations:: not specified
Details on mating procedure:: - Impregnation procedure: artificial insemination
Duration of treatment / exposure:: From day 6 to day 18 (at 200 mg/kg) and day 6 to day 13 ( 250 mg/kg)
Frequency of treatment:: Daily
Duration of test:: 30 days.
Dose / conc.:: 200 mg/kg bw/day
Remarks:: 153 mg/kg bw/day as salicylic acid (SA)
Dose / conc.:: 250 mg/kg bw/day
Remarks:: 192 mg/kg bw/day as SA
No. of animals per sex per dose:: 3 females (200 mg/kg)
4 females (250 mg/kg)
Control animals:: yes
Details on study design:: no data
Maternal examinations:: CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: No data

POST-MORTEM EXAMINATIONS: No data
- Sacrifice on gestation day 30
- Organs examined: none
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
Fetal examinations:: - External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:: no statistic were performed
Indices:: no data
Historical control data:: no
Details on maternal toxic effects:: Maternal toxic effects:yes

Details on maternal toxic effects:
In the dams given aspirin, severe food intake depression and weight loss occured during the dosing period. Further, 2 does treated with 200 mg/kg became moribund on day 20 and day 26 of gestation, respectively. Two does receiving 250 mg/kg were moribund early in gestation (day 13) and were sacrificed.
Dose descriptor:: NOAEL
Effect level:: < 200 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
All moribund females treated with 200 mg/kg had resorption of all embryos. The dams receiving 250 mg/kg had also resorbing embryos. At term, there was a marked reduction in litter size of dams receiving 200 mg/kg but no other effect in the several parameters evaluated. A single kit of a dam receiving 250 mg/kg aspirin, had hydrocephaly. There were no skeletal malformations among those examined, but the limited number (9) could preclude finding such defects.
Dose descriptor:: NOAEL
Effect level:: < 200 mg/kg bw/day
Basis for effect level:: other: fetotoxicity
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: Under the conditions of this test, aspirin induced maternal toxicity and fetotoxicity.
Executive summary:: In a reproduction study (schardein et al., 1969), aspirin was given to pregnant rabbits by gavage from day 6 through day 18 of gestation (200 mg/kg) or from day 6 through day 13 of pregnancy (250 mg/kg). All dams were sacrificed on day 30, one day prior to expected parturition. Then, each litter was examined for malformation of the skeleton and for determination of internal gross abnormalities. Severe food intake depression and weight loss occured during the dosing period in pregnant rabits given aspirin. Further, two does treated with 200 mg/kg became moribund on day 20 and day 26 of gestation, respectively, at sacrifice both had resorption of all embryos. Two does receiving 250 mg/kg were moribund early in gestation (day 13) and were sacrificed. They, likewise, had resorbing embryos. At term, there was a market reduction in litter size of dams receiving 200 mg/kg but no other effect in the several parameter evaluated. A single kit of a dam receiving 250 mg/kg aspirin had hydrocephaly. They were no skeletal malformations among those examined, but the limited number (9) could preclude finding such defects. There were no significant findings in kits of the control dams. Under the conditions of this test, aspirin induced maternal toxicity and fetotoxicity.

Reference 4

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: data not availalbe
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: NO GLP. Purity of the studied products not stated. Study well described.
Reason / purpose for cross-reference:: reference to other study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:: no
GLP compliance:: no
Limit test:: no
Species:: rat
Strain:: other: Holtzman
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: data not availalbe
- Age at study initiation: data not availalbe
- Weight at study initiation: data not availalbe
- Fasting period before study: data not availalbe
- Housing: data not availalbe
- Diet : ad libitum, Breeder's Ration (Parke, Davis& Company, Deroit, Michigan)
- Water : ad libitum
- Acclimation period: data not available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/- 1°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod : 12 hrs dark / 12 hrs light

IN-LIFE DATES: no data
Route of administration:: other: oral feed or gavage
Vehicle:: not specified
Details on exposure:: Data not available
Analytical verification of doses or concentrations:: not specified
Details on mating procedure:: - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: data not available
- Length of cohabitation: female and male rats were placed together until at least 10 females were inseminated
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:: day 6 through 15 of gestation
Frequency of treatment:: daily
Duration of test:: 21 days (day 0 throuh day 21 of gestation)
Dose / conc.:: 99 mg/kg bw/day
Remarks:: 2000 ppm in the diet (equivalent to 76 mg/kg bw/day as salicylic acid (SA))
Dose / conc.:: 224 mg/kg bw/day
Remarks:: 4000 ppm in the diet (equivalent to 172 mg/kg bw/day as SA)
Dose / conc.:: 250 mg/kg bw/day
Remarks:: by gavage (equivalent to 192 mg/kg bw/day as SA)
No. of animals per sex per dose:: 8-9 female per dose
Control animals:: yes, concurrent no treatment
Details on study design:: Sex: male/female
Duration of test: From day 6 to day 15 of gestation. Sacrifice on day 21 (1 day prior parturition)
Maternal examinations:: no data available
Ovaries and uterine content:: no data available
Fetal examinations:: - External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: No data
Statistics:: No
Indices:: no data available
Historical control data:: No
Details on maternal toxic effects:: Maternal toxic effects:yes

Details on maternal toxic effects:
The dams receiving aspirin had a decrease in food intake and weight gain. Those receiving 99 mg/kg had a 29% food intake depression with a 52% weight gain depression while those given 224 mg/kg had a 17 % food intake depression and a 90 % weight gain depression.
Dose descriptor:: NOAEL
Effect level:: < 99 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
In dams receiving the higher doses (224 or 250 mg/kg) of acetylsalicylic acid (aspirin), all pregnancies resulted in resorption. Those given 99 mg/kg showed no adverse effect, the reproductive parameters being comparable to the controls, and there were no gross defects in the pups. However, a number ofskeletal malformation appeared among the 53 pups examined. (See "in remarks on results" to obtain details on skeletal malformations)
Dose descriptor:: NOAEL
Effect level:: < 99 mg/kg bw/day
Basis for effect level:: other: teratogenicity
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: Under the conditions of this experiment, acetylsalicylic acid (aspirin) was embryotoxic at higher doses, and induced severe
alteration in the reproductive process and skeletal malformations of the offspring even at low doses.
Executive summary:: In a reproduction study (schardein et al., 1969), acetylsalicylic acid (aspirin) was given to pregnant rats in the diet on days 6 through 15 of gestation. Because of the poor conception rate and the large number of resorbed fetuses in those fed aspirin, another group of females was given 250 mg/kg of aspirin by gavage, and another group was given aspirin at the reduce level of 0.2 % in the diet for the same period of time. The dams receiving aspirin had a decrease in food intake and weight gain. Those receiving 99 mg/kg had a 52 % weight gain depression while those given 224 mg/kg had a 17 % food intake depression and a 90 % weight gain depression. In those dams receiving the higher doses (224 or 250 mg/kg) of aspirin all pregnancies resulted in resorption. Those given 99 mg/kg showed no adverse effect, the reproductive parameters being comparable to the controls, and there were no gross defects in the pups. However, a number of skeletal malformations appeared among the 53 pups examined. There were no significant findings in the 131 pups of control dams examined. Under the conditions of this experiment, acetylsalicylic acid (aspirin) induced maternal toxicity and was embryotoxic at higher doses, and induced severe alteration in the reproductive process and skeletal malformations of the offspring even at low doses.

Reference 5

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: no data
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: The study is adequately described. The test conditions are reasonably in compliance with guidelines, however the treatment period did not cover the whole period of organogenesis. Not GLP
Reason / purpose for cross-reference:: reference to other study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:: yes
Remarks:: Exposure was limited to the period of organogenesis (GD 8-14 only)
GLP compliance:: no
Limit test:: no
Species:: rat
Strain:: Wistar
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Nihon Rat Co. Ltd., Tokyo
- Age at study initiation: > 12 weeks old
- Weight at study initiation: ca. 200 g

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 2
- Humidity (%): 50-60
Route of administration:: oral: feed
Vehicle:: unchanged (no vehicle)
Analytical verification of doses or concentrations:: not specified
Details on analytical verification of doses or concentrations:: No data
Details on mating procedure:: - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: overnight

- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:: 1 week (from the 8th to 14th day of gestation)
Frequency of treatment:: continuously (in the diet)
Duration of test:: Day zero of pregnancy to the 56th day after birth
No. of animals per sex per dose:: 20 females
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale: no data
Maternal examinations:: CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: dayly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, placenta
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:: - External examinations: Yes: [all per litter]
- Soft tissue examinations: No data
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data
Statistics:: Statistics were performed but no data on the type of test performed
Indices:: No
Historical control data:: No
Details on maternal toxic effects:: Maternal toxic effects:yes

Details on maternal toxic effects:
- Transient bodyweight loss at 0.4%
- Salivation, piloerection at 0.4%
Dose descriptor:: NOAEL
Effect level:: 0.2 other: %
Basis for effect level:: other: maternal toxicity
Dose descriptor:: NOAEL
Effect level:: 0.1 other: %
Basis for effect level:: other: developmental toxicity
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- High fetal mortality at 0.4% (no live fetuses in 9/15 dams examined)
- Significant growth retardation for fetuses at 0.2 and 0.4%
- Increased fetal abnormalities at 0.2 and 0.4%: internal organ (kidney) abnormalities at 0.4% and skeletal bone abnormalities (absence or fragmentation or conjugation of cervical bone) at 0.2 and 0.4%
- In postnatal phase, only 6 newborns alive from one dam, all died within one day after birth, at 0.4%
- Minor changes in cervical bones of pups at day 56 after birth at 0.2%
Dose descriptor:: NOAEL
Effect level:: 0.1 other: %
Basis for effect level:: other: fetotoxicity
Dose descriptor:: NOAEL
Effect level:: 0.1 other: %
Basis for effect level:: other: teratogenicity
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: This academic non-GLP compliant study illustrates the potential of salicylic acid to induce embryofetal toxicity at dose levels equal to or higher than 0.2% and malformations at the maternally toxic dose level of 0.4% following dietary administration in Wistar rats between days 8 and 14 of gestation.
Executive summary:: In a teratogenic study (Tanaka et al., 1973a), salicylic acid was administered to pregnant rats at levels of 0.06, 0.1, 0.2 and 0.4 % in diet from the 8th to 14th day of gestation. The animals employed were a closed colony of Wistar strain rat and 20 pregnant rats were used in each group. A temporary body weight loss with toxic symptoms such as salivation, piloerection following the administration was observed in the 0.4 % group, with high mortality and growth retardation in fetuses. No toxic signs were observed in the three lower dose groups. Significant growth retardation was noticed in fetuses of 0.2 % group. Anomalies occurred at the two higher doses, with a high frequency of complex anomalies (cranioschisis, myeloschisis, pes varus, oligodactyly, etc) at 0.4%. For the sub-groups of dams retained for parturition and lactation, the postnatal observations showed in 0.4 % group only 6 pups were born alive from one dam and all died within 1 day after birth. There were no marked difference in litter size and in weaning rate at 8 weeks after birth for the other dose groups. Dose-related foetal growth retardation was noted from 0.2%. In the postmortem examination of offspring terminated at 8 weeks after birth, neither external nor internal organ anomalies were found in any group, but minor changes in cervical bone occurred in some pups of 0.2 % group. Under the conditions of the present experiment, salicylic acid administered in the diet has an embryotoxic effect on rat, with evidence of malformations at maternally toxic doses.

Reference 6

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: no data
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: The study is adequately described. The test conditions are reasonably in compliance with guidelines, however the treatment period did not cover the whole period of organogenesis. Not GLP.
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:: yes
Remarks:: Exposure was limited to the period of organogenesis (GD 8-14 only)
GLP compliance:: no
Limit test:: no
Species:: rat
Strain:: Wistar
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Nihon Rat Co. Ltd., Tokyo
- Age at study initiation: > 12 weeks old
- Weight at study initiation: 200 g
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): Laboratory chow (NMF, manufactured by Oriental Yeast Co., Tokyo).
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 2
- Humidity (%): 55 +/- 5
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
Route of administration:: oral: gavage
Vehicle:: CMC (carboxymethyl cellulose)
Details on exposure:: VEHICLE
- Concentration in vehicle: 0.5%
No other data.
Analytical verification of doses or concentrations:: not specified
Details on analytical verification of doses or concentrations:: No data
Details on mating procedure:: - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: overnight

- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:: 1 week (from the 8th to 14th day of gestation)
Frequency of treatment:: once a day
Duration of test:: Day zero of pregnancy to the 56th day after birth
No. of animals per sex per dose:: 20 females
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: no data
Maternal examinations:: CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, placenta
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:: - External examinations: Yes: [all per litter]
- Soft tissue examinations: No data
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data
Statistics:: Statistics were performed but no data on the type of test performed
Indices:: No
Historical control data:: No
Details on maternal toxic effects:: Maternal toxic effects:yes

Details on maternal toxic effects:
- Marked bodyweight loss at 300 mg/kg of salicylic acid, followed by reduced bodyweight gain.
- Feed and water consumption decreased at 300 mg/kg , the Mortalities were 3.
- Salivation, piloerection at 300 mg/kg of salicylic acid.
- Decreased uterine weight was observed in animals of the 150 and 300 mg/kg dose groups as compared to controls.
Dose descriptor:: NOAEL
Effect level:: 150 mg/kg bw/day
Basis for effect level:: other: maternal toxicity
: Key result
Dose descriptor:: NOAEL
Effect level:: 75 mg/kg bw/day
Basis for effect level:: other: developmental toxicity
Dose descriptor:: LOAEL
Effect level:: 150 mg/kg bw/day
Basis for effect level:: other: developmental toxicity
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- The fetal mortality increased in parallel with the doses (no live fetuses were obtained in 300 mg/kg salicylic acid group).
- Litter size and neonatal body weight, body length and tail length were significantly decreased in the 150 mg/kg salicylic acid group. The number of implantation sites or placental remnants and also some resorptions in these high dose groups were observed.
- Increased fetal abnormalities at 150 mg/kg and 300 mg/kg: external anomalies (cranioschisis, excencephaly and pseudomacroglossia), internal organ (kidney) abnormalities at 150 mg/kg and skeletal bone abnormalities (absence or fragmentation or fusion of cervical bone) at 150 mg/kg.
- In postnatal phase:
- No live newborn were obtained in the 300 mg/kg salicylic acid groups.
- The offspring from animals of 150 mg/kg salicylic acid group had decreased body length and tail length compared to controls.
- The thyroid weight of male offspring from the 75 mg/kg group was significantly increased and the adrenal gland weight of male offspring from the 150 mg/kg group was significantly decreased compared to controls.
- The incidences of external organ, internal organ, an skeletal anomalies in offspring were 0%, 5.0% and 0% respectively for the 75 mg/kg group and 13.7%, 17.2% and 79.2% respectively, for the 150 mg/kg group .
: Key result
Dose descriptor:: NOAEL
Effect level:: 75 mg/kg bw/day
Basis for effect level:: other: teratogenicity
: Key result
Dose descriptor:: NOAEL
Effect level:: 75 mg/kg bw/day
Basis for effect level:: other: fetotoxicity
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: Under the test conditions, salicylic acid is embryotoxic in the rats and induces malformations at maternally toxic doses. The following NOAELs were identified: NOAEL (materna toxicityl): 150 mg/kg, NOAEL (malformations): 75 mg/kg and the NOAEL (fetotoxicity): 75 mg/kg.
Executive summary:: In a teratogenic study (Tanaka et al., 1973 b), pregnant rats received consecutive daily oral doses of salicylic acid at dose levels of 75, 150 and 300 mg/kg in a 0.5% solution of sodium carboxymethylcellulose from the 8th to 14th day of gestation. The animals employed were a closed colony of Wistar strain rat and 20 pregnant rats were used in each group. In 300 mg/kg groups of salicylic acid, the body weight gains were inhibited with the toxic symptoms such as salivation, piloerection, and some animals died within a few days after the beginning of the administration, and high fetal mortality prevailed. Decreased uterine weight was observed in animals of the 150 and 300 mg/kg dose groups as compared to controls; these groups had 25.7% and 100% fetal mortality, respectively. Litter size and neonatal body weight, body length, and tail length were significantly decreased in the 150 mg/kg dose group. The incidences of external, internal, and skeletal anomalies in offspring autopsied at the 56 th day were 1.8%, 0%, and 2.5%, respectively, for the 75 mg/kg group and 27.8%, 12.7%, and 65.7%, respectively; for the 150 mg/kg group. The offspring from animals of 150 mg/kg salicylic acid group had decreased body length and tail lenfth compared to controls. The thyroid weight of male offspring from the 75 mg/kg group was significantly decreased compared to controls. The incidences of external organ, internal organ, and skeletal anomalies in offspring were 0%, 5.0% and 0% respectively, for the 75 mg/kg group and 13.7%, 17.2% and 79.2% respectively, for the 150 mg/kg group. The NOAEL (maternal): 150 mg/kg and the NOAEL (development): 75 mg/kg were identified. Under the conditions of the present experiment, salicylic acid administered by gavage is embryotoxic in the rats and induces malformations at maternally toxic doses.The teratogenic effect of salicylic acid may be considered as possibly due to direct action of the agent on the fetus, since a relative distribution of the agent was found in the fetus through the placental barrier.

Reference 7

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: supporting study
Study period:: no data
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: The protocol follows approximately the current guidelines. Not GLP-compliant
Reason / purpose for cross-reference:: reference to other study
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:: yes
Remarks:: Solely the period of organogenesis was examined
GLP compliance:: not specified
Limit test:: no
Species:: rat
Strain:: Sprague-Dawley
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: CLEA Japan Inc.
- Age at study initiation: ca. 9 weeks old
- Acclimation period: 4 to 6 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1
- Humidity (%): 55 +/- 5
Route of administration:: oral: gavage
Vehicle:: other: 0.5% methylcellulose in water
Details on exposure:: Administration volume = 5 mL/kg
Analytical verification of doses or concentrations:: not specified
Details on mating procedure:: - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: overnight (17 hr)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:: Gestation days 7 to 17
Frequency of treatment:: Once daily around 9.00 am
Dose / conc.:: 50 mg/kg bw/day
Remarks:: 38 mg/kg bw/day as salicylic acid (SA)
Dose / conc.:: 100 mg/kg bw/day
Remarks:: 77 mg/kg bw/day as SA
Dose / conc.:: 200 mg/kg bw/day
Remarks:: 153 mg/kg bw/day as SA
No. of animals per sex per dose:: 19 to 22 females
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale: no data
Maternal examinations:: CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: daily from 7 to day 20 of gestation

POST-MORTEM EXAMINATIONS: No data
- Sacrifice on gestation day 20
- Organs examined: none
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:: - External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:: Data obtained were statistically analysed by Wilcoxon's Rank Sum Test.
Indices:: no data
Historical control data:: No data
Details on maternal toxic effects:: Maternal toxic effects:yes

Details on maternal toxic effects:
Significantly decreased bodyweight at 200 mg/kg (aspirin) thoughout treatment and post-treatment periods.
Dose descriptor:: NOAEL
Remarks:: Aspirin
Effect level:: 100 mg/kg bw/day (nominal)
Basis for effect level:: other: maternal toxicity
Details on embryotoxic / teratogenic effects:: Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
In aspirin-treated groups only:
- Increased number of fetal resorptions and malformed survivors at 200 mg/kg
- Dose-dependently decreased fetal bodyweight at 100 and 200 mg/kg
- 21 fetuses (8.5%) presenting gross malformations at 200 mg/kg
- Significantly delayed ossification at 200 mg/kg
- Increased frequency of skeletal malformations (mainly absence, fusion, fragmentation or deformation of vertebral and costal bones) or variations at 200 mg/kg
Dose descriptor:: NOAEL
Remarks:: Aspirin
Effect level:: 100 mg/kg bw/day (nominal)
Basis for effect level:: other: teratogenicity
Dose descriptor:: NOAEL
Remarks:: Aspirin
Effect level:: 50 mg/kg bw/day (nominal)
Basis for effect level:: other: fetotoxicity
Abnormalities:: not specified
Developmental effects observed:: not specified
Conclusions:: This comparative teratogenicity study on two salicylic acid derivatives, proposed as anti-inflammatory agents (aspirin and diflunisal), in Sprague-Dawley rats illustrates the embryo-fetotoxic effects of aspirin at 100 or 200 mg/kg and malformations at 200 mg/kg, which are attributed by the authors to salicylic acid, as an hydrolysis metabolite able to cross placental barrier and result in significant fetal concentrations.
Executive summary:: In a teratogenicity study (Nakatsuka and Fujii, 1979), nulliparous Sprague-Dawley rats about 9 weeks of age were orally treated daily with aspirin at 50, 100 or 200 mg/kg from day 7 to day 17 of gestation. Several adverse effects were observed among the fetuses in the aspirin-treated groups. At 200 mg/kg the number of resorptions and malformed survivors were significantly increased. At 100 and 200 mg/kg the average body weights were significantly reduced in a dose-related manner. Skeletal examination revealed significant delay of ossification among the fetuses in aspirin-treated groups and the frequency of skeletal malformations or variations was significantly increased over control values in the 200 mg/kg group.

Reference 8

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

data not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP stated. Purity unknowm. But the protocol was close to the current guidelines.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

all recommended observations were not performed

GLP compliance:

not specified

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: about 2 months old
- Weight at study initiation: 200 g
- Fasting period before study: data not available
- Housing: 3-5 per cage
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: data not available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): data not available
- Air changes (per hr): data not available
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES:data not available

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not available
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug or sperm in vaginal smear] referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6 through to day 15 of pregnancy

Frequency of treatment:

single daily doses

Duration of test:

around the time of gestation (21 days)

Dose / conc.:

30 mg/kg bw/day

Remarks:

26 mg/kg bw/day as salicylic acid (SA)

Dose / conc.:

90 mg/kg bw/day

Remarks:

78 mg/kg bw/day as salicylic acid (SA)

Dose / conc.:

180 mg/kg bw/day

Remarks:

157 mg/kg bw/day as salicylic acid (SA)

No. of animals per sex per dose:

17-19 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: Examination of dams and fetuses made on day 21 of the pregnancy

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: every day

POST-MORTEM EXAMINATIONS: No data

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes:1/3 per litter
- Skeletal examinations: Yes: 2/3 per litter

Statistics:

Chi 2 test or t test were used.

Indices:

no data

Historical control data:

Historical control population (untreated rats) were given. Data were collected over the period of 63 months and based upon 750 litters.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
The females of the 180-mg/kg dose group reacted to the treatment by some reduction in food consumption.

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
In the 180 mg/kg group, the embryo- and fetolethality were markedly increased. In this group, 30% of the fetuses were malformed, the most common malformation being cranio(rachi)schisis (22.7% of the fetuses). A dose-related delay in growth was significant at 80 and 180 mg/kg bw, indicated by a diminished body weight and a retarded skeletal maturation.

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1 : Sodium salicylate: embryotoxicity and teratogenicity following maternal oral treatment on days 6 -15 of pregnancy.

dosage mg/kg	number of females with implantation sites	mean of implantation sites per female	number of females with abortions	embryo and fetolethality, mean % per dam	Number of live fetuses		mean body weight of live fetuses,g
dosage mg/kg	number of females with implantation sites	mean of implantation sites per female	number of females with abortions	embryo and fetolethality, mean % per dam	males	females	mean body weight of live fetuses,g
0	19	14.8	0/19	4.6	127	141	5.41 +/-0.42
30	17	15.4	0/17	5.0	121	128	5.42 +/-0.45
90	19	16.1	0/19	4.9	154	136	5.25 +/-0.41**
180	17	15.1	1/17	29.0*	93	88	3.86 +/-0.67**

* Chi2 test, p<0.01.

** t test, one-tailed, p<0.01.

Table2: sodium salicylate: delay of skeletal maturation as stated for the near-term fetuses

		dosage
		0	30 mg:kg	90 mg/kg	180 mg/kg
number of skeletons examined		180	165	192	123
ossification still absent in phalangeal nuclei, %	fore-limb	5.6	7.9	26.0a	93.5a
ossification still absent in phalangeal nuclei, %	hind-limb	43.9	41.2	77.6a	100a
5th sternebra stil incompletely ossified, %		6.7	8.5	5.7	78.9a
some centres of thoracic vertebrae dumbbell-shaped, %		1.1	1.2	10.9a	45.5a
some thoracic vertebrae with 2 small ossified centres, %		0	0	4.7a	83.7a

^a outside 99% confidence limits of control incidences

Conclusions:

Under the conditions of this test, sodium salicylate (NaS) was teratogenic in the rat following maternal peroral treatment during embryonic organogenesis.

Executive summary:

The effects of salicylic acid, acetylsalicylic acid or sodium salicylate on organogenesis have been investigated in a large number of studies in several animal species, using a variety of protocols. Many are mechanistic studies, using a single, often high, dose on a restricted number of gestation days. Relatively few are comparable to the prenatal developmental toxicity study OECD guideline 414. For Salicylic acid (SA), two studies in rat (Tanaka et al, 1973a and Tanaka et al 1973b) are acceptable as key studies, although SA was administered only from GD8 to GD14. To complement these studies and to provide data on developmental toxicity in the rabbit, two recent developmental toxicity studies on Acetylsalicylic acid (ASA, aspirin) in rats (Gupta et al, 2003) and rabbits (Cappon et al, 2003) have been included as key studies. These studies complied with current ICHguidelines for pharmaceuticals.

In a teratogenicity study (Tanaka et al., 1973a), salicylic acid was administered to pregnant Wistar rats at levels of 0.06,0.1, 0.2 and 0.4 % in the diet (30, 50, 100, 200 mg/kg bw/day) on GD 8-14. The high dose of 0.4% caused maternal toxicity, high foetal mortality, growth retardation and a high frequency of complex anomalies including cranioschisis, myeloschisis, pes varus, oligodactyly. At 0.2%, significant foetal growth retardation and a low frequency of anomalies. No effect levels were NOAEL (maternal): 0.2% (100 mg/kg bw/day) and NOAEL (development): 0.1% (50 mg/kg bw/day).A parallel study by gavage (Tanaka, 1973b) at 75, 150 and 300 mg/kg bw gave similar results, with no effect levels NOAEL (maternal): 150 mg/kg and NOAEL (development): 75 mg/kg bw.

In a clinical segment II study, ASA was administered by oral gavage to pregnant Sprague-Dawley rats at 50, 125 or 250 mg/kg bw/day (equivalent to 38, 96, 192 mg/kg bw as SA) during organogenesis (GD6 -17) (Gupta & al, 2003). There was a dose-related reduction in maternal bodyweight gain, significant in the mid and high dose groups. At 250 mg/kg bw/day, ASA induced increases in early resorptions, increased post-implantation loss, increased variations and malformations. At 125 mg/kg, foetal viability was reduced.

A number of valid supporting studies in rats report similar results to those described in the key rat studies above. Fritz and Giese (1990), showed a marked increase in embryonic and foetal mortality, delayed ossification and malformations at 180 mg/kg NaS on GD 6-15. Nakatsuka and Fujii (1979), treated SD rats with ASA on GD 7-17. At 200 mg/kg the number of resorptions and malformed survivors were significantly increased. At 100 and 200 mg/kg the average body weights were significantly reduced in a dose-related manner. Schardein et al. (1969) showed ASA to be embryotoxic to rats fed doses of 250 mg/kg bw/day by gavage, or 0.2 or 0.4% (99 or 240 mg/kg bw/day) in the diet on DG 6-15.. These doses caused significant reduction in maternal bodyweight gain. At 224 or 250 mg/kg ASA, all pups were resorbed. There were a number of skeletal malformations in the pups at 99 mg/kg bw/day.

The results of the key and supporting studies in rats demonstrate that SA has an embryofoetotoxic effect in rats at doses not causing clear maternal toxicity, with evidence of malformations at maternally toxic doses.

ASA was administered by oral gavage to pregnant New Zealand White rabbits at 125, 250 or 350 mg/kg bw/day on GD7-19 (Cappon & al, 2003). Maternal body weight gain was significantly reduced in the mid and high dose groups from GD7 to GD13. Food consumption was also reduced in these groups. Three high dose does and one mid dose doe died during the study. There were no treatment-related effects on corpora lutea, implantation sites, pre-implantation losses or embryofoetal mortality. There were no treatment-related visceral or external anomalies. Reduction in mean foetal weight at 350 mg/kg bw/day was the only developmental adverse effect reported at this maternally toxic dose.

In a supporting study (Schardein et al, 1969), rabbits received ASA at 200 or 250 mg/kg on DG 6-13 or GD6-18. ASA induced maternal toxicity but no skeletal malformations or other effects on offspring.

It is clear that there are differences in sensitivity between the tested species. From developmental toxicity studies equivalent to OECD guideline 414, the rabbit is seen to be considerably less sensitive than the rat to the developmental toxicity of SA and other salicylates. In the multi-generation studies equivalent to OECD guideline 416, it was also seen that the mouse was less sensitive than the rat in this respect.

The rat data provide a “worst case” for risk assessment, however there is a considerable body of published data on the effects of ASA administered during human pregnancy which indicate that pregnant women and their offspring are relatively insensitive to ASA developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
: 75 mg/kg bw/day
Species:: rat

Additional information

Developmental effects: animal data

The developmental effects presented are from studies with salicylic acid, ASA or NaS.

Studies in Rats

In a pre-natal developmental toxicity study (Tanaka et al., 1973a, reliability 2), salicylic acid was administered to pregnant Wistar rats at levels of 0.06%, 0.1%, 0.2% and 0.4% in the diet (50.7 +/- 0.6, 77.4 +/- 1.0, 165 +/- 2.1, 205.9 +/- 18.9 mg/kg bw/d, respectively) on gestation days (GD) 8-14. The two lower doses (i.e. 50.7 and 77.4 mg/kg bw/d) caused neither maternal nor foetal effects. A marked body weight loss of dams was observed in the 0.4% group at the beginning of salicylic acid administration, but a gradual increase in bw was then observed after GD 11 day. This decrease in bw was assumed to be due to a decrease in food intake, but no deaths were observed. No marked changes were noticed in other groups. Very low uterine weights of foetuses and significantly lower placental weights were obtained in the 0.4% group, but there were no marked differences in the number of corpora lutea or in the rate of nidation in all groups. The dose of 0.2% (165 mg/kg bw/d) caused foetal effects (foetal anomalies and growth retardation) in the absence of maternal effects. This dose resulted in a maternal serum concentration of about 116 microgram/mL. The highest dose of 0.4% (205.9 mg/kg bw/d) induced maternal effects expressed as temporary body weight loss with toxic symptoms (salivation, piloerection) and the following foetal effects: high fetal mortality (no live foetuses in 9/15 dams examined), high frequency of complex anomalies (cranioschisis, myeloschisis, pes varus, oligodactyly etc.) and dose-related foetal growth retardation. At the dose of 0.2%, the body weight and length and the tail length were statistically significantly decreased. At the dose of 0.4% litter size and body weight and length as well as tail length were statistically significantly decreased.

The derived no adverse effect levels (NOAELs) were: maternal - 0.2% (165 mg/kg bw/d) and developmental - 0.1% (77.4 mg/kg bw/d).

A parallel study by gavage (Tanaka, 1973b) at 75, 150 and 300 mg/kg bw/d gave similar results, with NOAEL (maternal) of 150 mg/kg bw/d and NOAEL (development) of 75 mg/kg bw/d.

In an experimental segment II study (Gupta et al., 2003, Klimisch score 1), ASA was administered by oral gavage both in single and multiple dose studies.

In the single dose study the dosage was as follows: GD9 (0, 250, 500 and 625 mg/kg bw), GD10 (0, 500, 625 and 750 mg/kg bw), GD11 (500, 750 and 1000 mg/kg bw). No maternal deaths were noted in any dose group; there were no treatment related clinical signs or necropsy findings. Dose-dependent decreases in body weight gain and food consumption were observed for dams dosed on the days when treatment was administered. Body weight gains were decreased for the duration of the study, whereas the food consumption remained decreased for 2 days after the administration of ASA. The decrease in body weight gain was only partly due to reductions in food consumption as the magnitude of this change was minimal compared with the body weight gain decrements. The decrease in foetal weight and the number of foetuses contributed to the decrease in body weight gain. A dose-dependent decrease in the number of viable foetuses was observed in dams administrated ASA on each day with the exception of 500 mg/kg dose group on GD11 although the decreases were not always statistically significant. An increase in resorptions with an associated increase in post-implantation loss was observed in the mid- and high-dose groups on GD10 and GD11. The number of viable foetuses decreased in almost all dose groups, with accompanying decreases in uterine weight in all dose groups on GD9 and GD11 and in the mid- and high-dose groups on GD10. Significant increases in malformations were reported only from 500 mg/kg for GD9 administration or from 625 mg/kg for GD10 or GD11.

In the multiple dose study the dosage was of 0, 50 mg/kg bw/d (38 mg/kg bw/d as salicylic acid), 125 mg/kg bw/d (96 mg/kg bw/d as salicylic acid), 250 mg/kg bw/d (192 mg/kg bw/d as salicylic acid). Maternal toxicity was reported as a dose-dependent decrease in maternal body weight that was statistically significant at the mid-dose (85% of control) and high-dose (52% of control). Hence, the decreases in food consumption were partly responsible for the decrease in body weight. Irregular respiration and sporadic salivation were noted in the dams at 250 mg/kg bw/d. One dam given 125 mg/kg bw/d was killed as moribund on GD13 due to severe weight loss and associated inappetence. At necropsy, this dam showed no unusual findings and no other mortality was noted. The foetal toxicity was expressed as a dose-dependent decrease in the number of foetuses. This was due to the dose-dependent decrease in the numbers of viable foetuses across the dose group. The malformations were statistically significantly increased only in the high dose (250 mg/kg) group and included ablepharia, craniorachischisis, bent forepaw, kinked tail, protruding tongue, gastroschisis, ectopic adrenal, ventricular septal defect (VSD), diaphragmatic hernia, hypoplastic kidney and hypoplastic testis.

Fritz and Giese (1990, Klimisch score 2) performed a gavage study on 17-19 females per dose with NaS during GD 6-15. The dosage was 30, 60 and 90 mg/kg bw/d. The maternal toxicity was seen only at the highest dose and was described as some reduction in food consumption. The foetal toxicity could be observed at the mid dose (in the absence of maternal toxicity) as a dose-related delay in growth. At the highest dose the foetal toxicity was described as a dose-related delay in growth and malformations in 30% of the foetuses, the most common malformation being cranio(rachi)schisis (22.7% of the foetuses).

Nakatsuka and Fujii (1979, Klimisch score 2) treated SD rats with ASA on GD 7-17 with 3 doses: 50, 100 and 200 mg/kg bw/d. Neither maternal nor foetal toxicity were present at the lowest dose. At the middle dose, dose-dependently decreased foetal bodyweight was described in the absence of maternal toxicity. At 200 mg/kg bw/d maternal toxicity was described as significantly decreased bodyweight and the foetal toxicity as: increased number of foetal resorptions and malformed survivors, dose-dependently decreased foetal bodyweight, 21 foetuses (8.5%) with gross malformations, significantly delayed ossification and increased frequency of skeletal malformations (mainly absence, fusion, fragmentation or deformation of vertebral and costal bones) or variations.

Schardein et al. (1969, Klimisch score 2) treated rats with ASA at doses of 99 mg/kg bw/d (0.2 % in diet), 224 mg/kg bw/d (0.4% in diet) and 250 mg/kg bw/d (by gavage) during GD 6-15. Maternal toxicity was reported at 0.2% in diet (29% food intake depression with a 52% weight gain depression) and 0.4% in diet (17 % food intake depression and a 90% weight gain depression). Fetal toxicity was registered at all doses with skeletal malformations at 99 mg/kg bw/d and 100% resorptions at the two higher doses.

Studies in Rabbits

ASA was administered by oral gavage to pregnant New Zealand White (NZW) rabbits at 125, 250 or 350 mg/kg bw/d on GD 7-19 (Cappon et al., 2003, Klimisch score 2). Maternal body weight gain was significantly reduced in the mid and high dose groups from GD 7 to 13. Food consumption was also reduced in these groups. Three does given the high dose and one given the mid dose died during the study. There were no treatment-related effects on corpora lutea, implantation sites, pre-implantation losses or embryofoetal mortality. There were no treatment-related visceral or external anomalies. Reduction in mean foetal weight at 350 mg/kg bw/d was the only developmental adverse effect reported at this maternally toxic dose.

In a supporting study (Schardein et al., 1969, Klimisch score 2), rabbits received ASA at 200 or 250 mg/kg bw/d on GD 6-13 or GD 6-18. ASA induced maternal toxicity. A single kit of a dam had hydrocephaly. There were no skeletal malformations among those examined, but the limited number (9) could have precluded finding such defects. There were no significant findings in kits of the control dams. Under the conditions of this test, ASA induced maternal toxicity and foetotoxicity.

Studies in Monkeys

The study of Wilson et al. (1977) is not compliant with OECD TGs; its original purpose was to elucidate toxicokinetic aspects, namely the distribution and embryotoxicity of ASA in rats versus monkeys. Since the administration of ASA was performed during the organogenesis period, some conclusions may however be drawn. Unlike other studies, the protocol of administration was twice per day by gavage. For rats the doses were 100, 150, 175 and 200 mg/kg bw (twice daily on GD 9-12) and for monkeys 100 and 150 mg/kg bw (twice daily, for 10 days starting on GD 23). The same doses were given to non-pregnant females of both species for the purpose of determining comparative plasma concentrations.

Maternal toxicity in rats was described as occasional death and weight loss at 200 mg/kg bw twice daily. Foetal toxicity in rats showed significant effects on intrauterine death, growth and malformations rates at 150 mg/kg (twice daily). At this dose, the percentage of dead or resorbed foetuses was 34% and increased to 73% at the highest dose of 200 mg/kg bw twice daily. Also, the percentage of malformed survivors (including those with cardiac, facial, brain, spinal, tail and other skeletal defects) increased from 55% at 150 mg/kg to 100% at 200 mg/kg bw.

In monkeys, the number of aborted or resorbed foetuses was the same (3) at both dosages. At the dose of 100 mg/kg bw twice daily the foetal effects were reported as growth retardation and at 150 mg/kg bw (twice daily), growth retardation and malformations such as gross abnormality, cranioshisis and cystic kidney were reported. At both doses there were also foetuses which were observed to be normal. The conclusion was that 150 mg/kg twice daily is in the teratogenic range.

A comparison of serum concentration of salicylic acid in mothers vs. whole embryo concentration was performed. Unbound salicylate in rat plasma ranged from 30% to 50% of the total plasma concentration and was closely paralleled by the concentration in the rat embryo. Unbound salicylate in monkey plasma was lower, ranging from 17% to 30% of the total plasma concentration and was to some degree paralleled by the concentration in the monkey embryo. The greater embryotoxicity of ASA in the rat compared to the monkey correlated with higher concentrations and longer duration of concentrations in the respective embryos on a day-to-day basis. The general conclusion was that this association only partially explains the difference between species; the mode of action within the embryo must not be neglected.

Developmental effects: human information

Despite its long usage, data regarding human exposure to salicylic acid itself is lacking. To fill the information gap, an assessment was performed using human data on ASA.

The assessment of “Low doses” in pregnancy

The “low doses” in pregnancy are indicated for “prevention of multiple miscarriage, pregnancy-induced hypertension and other complications of pregnancy”. The (retrospective) cohort study performed by Bard (2012) was provided for this dose range. To further extend the analysis, the DS submitted an additional critical review by the same author (Bard 2015).

The aim of the study and the analysis was to address the effects of ASA within this dose range on the following endpoints: maternal bleeding, neonatal haemostatic abnormalities, pregnancy duration and labour, prevention of pre-eclampsia and intra-uterine foetal growth retardation, stillbirths and infant mortality, birth weight, birth defects and early childhood development. Particular aspects that raised concern were also analysed; the premature closure of Ductus arterious, the occurrence of gastroschisis and congenital cryptorchidism.

As a final conclusion of the study it was stated that: “no adverse effect of aspirin treatment can be considered as established, either at low (<150 mg daily) or higher, usual dose”. To further illustrate the overall conclusion with respect to dosages higher than that mentioned above, three epidemiological studies (Slone, 1976; Shapiro, 1976; Kozer, 2002) were cited; the conclusion was that the use of aspirin at up to the maximum recommended therapeutic dose of 4000 mg/d (equivalent to 66.7 mg/kg bw/d as ASA, or 51 mg/kg bw/d as salicylic acid) have largely demonstrated an absence of increased risk of adverse pregnancy outcome in terms of frequency of stillbirth, neonatal mortality, birth defects or developmental delay.

The assessment of “High dose” ASA as prescribed in pregnancy

As already stated, ASA is an NSAID which may be prescribed at “high dose” levels for long-term treatment of a number of severe inflammatory conditions. Only limited information is available regarding the effects of such prescribed medicinal usage of ASA during pregnancy.

In a retrospective survey of 103 patients taking high dose ASA (at least 3250 mg per day) for rheumatoid arthritis or other inflammatory conditions, Lewis and Schulman (1973) reported an increased mean gestational length and increased duration of labour. No malformations was reported, however the study covered ASA exposure only throughout “at least” the last six months of pregnancy, so it cannot be established how many of these patients were also exposed to ASA during the first trimester.

The study of Østensen & Østensen (1996) was not included in the analysis since it had no specific information related to ASA.

Overall, the available data regarding therapeutic doses over 3g/d do not show any association between ASA and malformations in humans.

RAC Opinion (2016)

In March 2016, the Committee for Risk Assessment of the European Chemical Agency proposed to classify salicylic acid as a category 2 reproductive toxicant (ECHA, 2016). The classification is based on adverse developmental effects in two animal species (rat and monkey).

SCCS Opinion (2018)

Following review of the available toxicology data, the pivotal study (for deriving the point of departure (POD) as a toxicological benchmark for the safety evaluation of salicylic acid) remains the same in this dossier as was concluded by the SCCNFP in 2002, namely the developmental toxicity study on salicylic acid by Tanaka et al., 1973a. The POD is expressed as a no observed adverse effect level (NOAEL) of 75 mg/kg/day relating to the most sensitive toxic endpoint i.e. teratogenicity in the rat as the most sensitive species.

Justification for classification or non-classification

Fertility:

Not classified for effects on reproduction (fertility) according to EU and GHS (UN/EU) criteria.

Based on the available studies, the RAC concluded in 2016 that there was insufficient evidence that salicylic acid exhibits adverse effects on sexual function and fertility and concluded that no classification for salicylic acid for adverse effects on sexual function and fertility was justified..

Development:

Taking into account the available data, including pharmacokinetics, in vitro tests with ASA and salicylic acid, developmental studies in animals (positive findings in rat and monkey studies and a negative rabbit study), human epidemiology and medical experience, the RAC considered classification of salicylic acid as Repr. 2; H361d (Suspected of damaging the unborn child) to be justified.

In its assessment and comparison with the criteria for the development of the offspring endpoint, RAC took into consideration the following:

- There is robust evidence of developmental effects in animals which justifies classification. In animals, the developmental toxicity was clearly shown in two out of three species. The pattern and magnitude of the effects shown in rats but also in monkeys are sufficient to presume that salicylic acid is a developmental toxicant and to justify classification in Category 1B;

- According to experts in the field of pharmaceuticals, ASA is not considered as being a major teratogen, but may have some potential for teratogenic effects, and it should be noted that prostaglandin inhibitors in general, including ASA, could have other adverse effects on foetuses, especially on their renal development and during the third trimester on the development of the circulatory system;

- However, neither ASA nor salicylic acid are proven human developmental toxicants. There is a lack of evidence to support an increased risk of birth defects following exposure to ASA. Also, the evidence for other developmental effects has uncertainties. Taking that into account, classification in Category 1A is not justified.

- Although the information on effects of ASA on development in humans at “high doses” is marginal, it should be acknowledged and cannot be discarded when discussing classification in Category 1B versus Category 2.

- It is noted that the available human epidemiological data on ASA was rather contradictory and with only a few reported exposures at higher doses, nevertheless demonstrated no clear evidence of malformations in humans. Hence, the RAC concluded that Category 1B may not be justified.

Additional information

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	% females (pregnancies/ matings)
	N (females mated twice	2/2	1/2	0/2
first generation
control	20	85,0	5,0	10,0
0,25%	22	72,3	27,3	0
0,50%	25	72,0	28,0	0
second generation
control	22	9,1	72,7	18,2
0,25%	18	16,7	66,7	16,6
0,50%	16	18,9	68,8	12,4

	total born	live born	not killed at birth	alive 5 days	weaned 21 days
first generation
control	12,51	12,06	12,03	11,23	9,04
0,25%	10,68	10,58	10,53	10,03	9,06
0,50%	10,56	10,3	10,19	9,42	8,96
second generation
control	9,8	9,5	9,5	8,25	6,09
0,25%	11,5	11,5	11,5	11,2	7,95
0,50%	9,82	9,82	9,71	9,71	8,89

	stillborn	viability	lactation	reproduction
first generation
control	3,65	93,1	80,5	75
0,25%	0,99	94,8	90,3	85,6
0,50%	2,42	91,4	95,2	87
second generation
control	3,06	86,8	73,8	64,1
0,25%	0	97,6	70,8	69,1
0,50%	0	98,8	91,6	90,5

% females (pregnancies/ matings)
	N (females mated twice)	2/2	1/2	0/2
first generation
control	25	60,0	32,0	8,0
0,25%	24	70,8	25,0	4,2
0,50%	23	60,9	17,4	21,7
second generation
control	28	39,3	39,3	21,4
0,25%	30	33,3	36,7	30,0
0,50%	30	33,3	43,3	23,4

	total born	live born	not killed at birth	alive 5 days	weaned 21 days
first generation
control	11,53	11,34	11,32	11,16	9,38
0,25%	10,48	10,38	10,33	10,23	8,85
0,50%	11,03	10,63	10,53	9,38	8,09
second generation
control	9,55	8,94	8,94	8,76	8,02
0,25%	7,1	6,94	6,94	6,81	5,81
0,50%	8,49	8,33	8,33	7,58	6,41

Treatment	mg/kg	sex treated	Pregnant/inseminated	Litter size	female pups	male pups	female weight	male weight	viability (%)
standard ration	0	Sire	10/10	8.9	4.5	4.4	5.3	5.6	88
standard ration	0	Dam	16/20	10.6	5.0	5.6	6.1	6.3	84
Aspirin	209	Sire	11/13	10.7	5.3	5.5	5.2	5.6	95
Aspirin	210	Dam	16/21	9.4*	0	0	0	0	0

Concentration of salicylic acid intake (%)	0	0.06	0.1	0.2	0.4
Daily salicylic acid intake (mg/kg)	0	50.7 +/- 0.6	77.4 +/- 1.0	165 +/- 2.1	205.9 +/- 18.9

	stillborn	viability	lactation	reproduction
first generation
control	1,6	98,4	84,1	82,7
0,25%	0,95	98,6	86,8	85,5
0,50%	3,68	88,2	86,3	76,2
second generation
control	6,35	98	91,6	89,8
0,25%	2,27	98,1	85,3	83,7
0,50%	1,08	90,9	84,6	76,9

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Salicylic acid

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

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Effect on fertility: via oral route

Effect on fertility: via inhalation route

Effect on fertility: via dermal route

Additional information

Effects on developmental toxicity

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Effect on developmental toxicity: via oral route

Additional information

Justification for classification or non-classification

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