Methyl salicylate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL for fertility in rat is 300 mg/kg/day (no adverse effect up to the highest dose level by subcutaneous route).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

unpublished report cited by FDA and RAC (ANSES has access to the original studies cited in the FDA report, and considers this study as the Key study with a Klimisch reliability of 1)

Principles of method if other than guideline:

Males were dosed daily 2 weeks prior to mating, throughout mating period and up to one day prior to euthanasia ((total of 52 days). Females were dosed daily 2 weeks prior to mating, throughout mating period and up to Gestation Day 6 (total of 30 days). Sacrifice of females on GD13.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crj:CD(SD)IGS rats

Sex:

male/female

Route of administration:

subcutaneous

Vehicle:

corn oil

Details on exposure:

dose volume of 1 mL/kg

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the dosing period and once daily the other periods

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: males BW were recorded twice weekly throughout the dosing period until euthanasia. Female BW were measured twice weekly from the start of the mating and daily during gestation.

FOOD CONSUMPTION : male's food consumption was recorded twice weekly throughout the dosing period until euthanasia. Female's food consumption was measured twice weekly from the start of dosing to the start of mating and daily during gestation.

WATER CONSUMPTION AND COMPOUND INTAKE: No data

Oestrous cyclicity (parental animals):

Vaginal smears for determining the stage of estrous were evaluated daily from the start of administration and continued until evidence of copulation was observed.

Sperm parameters (parental animals):

sperm mobility and morphology were analysed.

Postmortem examinations (parental animals):

GROSS NECROPSY
- Gross necropsy consisted of examination of the organs and tissues.

HISTOPATHOLOGY / ORGAN WEIGHTS
The testes, left epididymis, ovaries and skin of the treated site were preserved for histological examination. Also organs with lesions were preserved for histological examination.
Weights of the following organs were obtained: testes, epididimis.

One male at 300 mg/kg/day showed hypoactivity, bradypnea, hypothermia and blanching on day 3 and died on day 4. Crust on the treated site and/or loss of hair were observed in 2 females at 300 mg/kg/day from day 9 of administration to day 13 of gestation. A significant lower body weight, body weight gain and food consumption was observed in males and females at the highest dose. There was no significant difference in the weights of the testes or epididymides. There was no significant difference in the count of oestus or estrous cycle. The copulation indices were 100, 100, 95.00, 94.74% for each group, respectively. The male and female fertility indices were 100, 90.00, 94.74, 94.44% for control, 30, 100 and 300 mg/kg/day respectively. There was no significant difference between control and methyl salicylate groups in the sperm form anomalies index, sperm count or sperm motility. There was no significant difference in the numbers of implants or live embryos, pre-implant low index or dead embryo index. A significant decrease in the number of corporea lutea was observed at 100 mg/kg/day (1.84% versus 4.81% in control) but not at 300 mg/kg/day (3.25%). Plasma salicylic acid concentration was measured on day 0 and day 13 of administration. The increase was nearly dependent on increases in the dose ratio and was scarcely affected by repeated dosing. No sexual difference was observed.

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: no effect observed on fertility

Remarks on result:

not measured/tested

Reproductive effects observed:

no

Executive summary:

In a study summarized in FDA (2006) and RAC opinion (2019), 20/sex rats were exposed subcutaneously to methyl salicylate (MeS) at 0, 30, 100 or 300 mg/kg/day 2 weeks prior to mating until sacrifice of males and until gestation day 6 for females. Females were sacrificed on gestation day 13. One male at 300 mg/kg/day showed hypoactivity, bradypnea, hypothermia and blanching on day 3 and died on day 4. Crust on the treated site and/or loss of hair were observed in 2 females at 300 mg/kg/day from day 9 of administration to day 13 of gestation. A significant lower body weight, body weight gain and food consumption was observed in males and females at the highest dose. There was no significant difference in the weights of the testes or epididymides. There was no significant difference in the count of oestus or estrous cycle. The copulation indices were 100, 100, 95.00, 94.74% for each group, respectively. The male and female fertility indices were 100, 90.00, 94.74, 94.44% for control, 30, 100 and 300 mg/kg/day respectively. There was no significant difference between control and methyl salicylate groups in the sperm form anomalies index, sperm count or sperm motility. There was no significant difference in the numbers of implants or live embryos, pre-implant low index or dead embryo index. A significant decrease in the number of corporea lutea was observed at 100 mg/kg/day (1.84% versus 4.81% in control) but not at 300 mg/kg/day (3.25%). Plasma salicylic acid concentration was measured on day 0 and day 13 of administration. The increase was nearly dependent on increases in the dose ratio and was scarcely affected by repeated dosing. No sexual difference was observed. In conclusion, the NOAEL for general toxicity is 100 mg/kg/day and the NOAEL for fertility and early development was 300 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Results supported by several reliable other studies on the substance itself.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Summary of the data as reported in the CLH dossier and RAC opinion:

In a study (summarized in FDA (2006a)), 20/sex rats were exposed subcutaneously to methyl salicylate (MeS) at 0, 30, 100 or 300 mg/kg/day 2 weeks prior to mating until sacrifice of males and until gestation day 6 for females. Females were sacrificed on gestation day 13. One male at 300 mg/kg/day showed hypoactivity, bradypnea, hypothermia and blanching on day 3 and died on day 4. Crust on the treated site and/or loss of hair were observed in 2 females at 300 mg/kg/day from day 9 of administration to day 13 of gestation. A significant lower body weight, body weight gain and food consumption was observed in males and females at the highest dose. There was no significant difference in the weights of the testes or epididymides. There was no significant difference in the count of oestus or estrous cycle. The copulation indices were 100, 100, 95.00, 94.74% for each group, respectively. The male and female fertility indices were 100, 90.00, 94.74, 94.44% for control, 30, 100 and 300 mg/kg/day respectively. There was no significant difference between control and methyl salicylate groups in the sperm form anomalies index, sperm count or sperm motility. There was no significant difference in the numbers of implants or live embryos, pre-implant low index or dead embryo index. A significant decrease in the number of corporea lutea was observed at 100 mg/kg/day (1.84% versus 4.81% in control) but not at 300 mg/kg/day (3.25%). Plasma salicylic acid concentration was measured on day 0 and day 13 of administration. The increase was nearly dependent on increases in the dose ratio and was scarcely affected by repeated dosing. No sexual difference was observed. In conclusion, the NOAEL for general toxicity is 100 mg/kg/day and the NOAEL for fertility and early development was 300 mg/kg/day.

Two studies have been conducted on MeS in CD-1 mice by gavage according to the NTP continuous breeding protocol (NTP, 1984a, 1984b).

In the first study (NTP, 1984a), male and female mice were exposed to 25, 50 and 100 mg/kg bw/day of MeS for 7 days prior to mating, during 98 days of cohabitation (allowing the production of about 4 litters) and then during a separation period of 21 days during which final litters were delivered (task 2). A second generation was then produced only for the highest dose group (task 4): the mothers were dosed through weaning and F1 mice were dosed until mated at about 74 days of age. Examinations were rather limited in parental animals (clinical signs and body weight, sperm measures (F1), fertility and mating index, limited examination of organ weight, gross and histopathology) and offsprings (number, sex, live and dead, body weight). There was no treatment related effect on parental survival, body weight and food consumption. No adverse effects were reported on fertility, number of pups per litter, percentages of live pups or pup weight. Necropsy of F1 mice revealed no adverse effects on body or organ weights or sperm motility, density or morphology. In task 4, mating and fertility indices were decreased at 100 mg/kg bw/day but it was not statistically significant (76% vs 95% for mating index and 65% vs 89% for fertility index). Based on the absence of statistically significant effect on fertility and development, the NOAEL were set at 100 mg/kg bw/day. It should be noted that this study does not permit a full assessment of reproductive and developmental toxicity considering the limited numbers of parameters assessed in this study compared to OECD test guidelines.

In the second NTP study (NTP, 1984b), male and female mice were exposed to 100, 250 and 500 mg/kg bw/day of MeS for 7 days prior to mating, during 100 days of cohabitation (allowing the production of about 4 litters) and then during a separation period of 21 days during which final litters were delivered (task 2). Examinations were rather limited: only clinical signs in parents, fertility index, number of litter produced, number of live/died pups and body weight were reported. No effect on fertility index (number of fertile/cohabite x 100) was observed (94-100%). No treatment related effect on parental mortality, clinical signs and body weight was reported. Reduced pup viability was reported at the high dose with decrease in the mean number of litter, in the average of live pups per litter and the proportion of pups born alive. At 250 mg/kg bw/day, a reduction in pup weight (about -4%) was reported in females. Based on the absence of effect on fertility index, the NOAEL for reproduction was 500 mg/kg bw/day. The NOAEL for development was 100 mg/kg bw/day based on a decrease of pup body weight. In order to discriminate which sex (or sexes) may be affected by the chemical exposure, a cross-over mating trial (task 3) was carried out where high-dose animals of each sex were mated to control mice of the opposite sex. An affected sex cannot be determined due to fertility problem in the control groups (29% in the first task 3 and 41% in the second task 3 versus 41-72% in the treated groups). It should be noted that this study does not permit a full assessment of reproductive and developmental toxicity considering the limited numbers of parameters assessed in this study compared to OECD test guidelines.

In a 3-generation study (Collins et al., 1971), methyl salicylate (MeS) was administered to male and female Osborne-Mendel rats in the diet at 500, 1500, 3000 and 5000 ppm (equivalent to 25, 75, 150, 250 mg/kg bw as MeS). Parental generation rats were fed MeS for 100 days prior to mating, then throughout two mating, gestation and lactation periods (until weaning of the F3 offspring). Each generation of rats was mated twice. Examinations performed in this study were very limited and consisted on fertility index, litter size, viability at birth, on day 4 and at weaning, external examination of newborn and weanling rats (all generations, all matings), histopathological examination of liver and kidney (for the 3rd generation only). No examination of reproductive tract (including histopathology, sperm and oestrus cycle measures…) was performed in both parents and offspring animals. Furthermore, peri- and post-natal development (including functional development, sexual maturation…) were not assessed. No significant effect was reported in fertility index at any dose for any generation. According to the authors, “appreciable decreases can be seen, however, in the second and third generation matings at 5000 ppm level”. Indeed, fertility indices (number of litters casts/number of females exposed to mating) were 85% and 77% for the first and second matings of the 2nd generation at 5000 ppm versus 100% in control. In the third generation, fertility indices were 89% vs 100% in the first mating and 84% vs 90% in the second mating. Adverse effects were reported on offspring, such as decreases in average litter size, number of liveborn progeny per female, viability (liveborn), survival (survivors on day 4) and weaning survival. These effects are only statistically significant in the 2nd generation, with a dose-related decrease starting from 1500 ppm. Decreases in weight at the weaning appeared consistently from 3000 ppm. There was no external abnormality or histopathological effect on the liver and kidney of offspring of the 3rd generation at weaning. Based on the absence of statistically significant effect on fertility, the NOAEL for fertility was set at 250 mg/kg bw/day. The NOAEL for development was 75 mg/kg bw/day based on pup mortality and decreased weight. It should be noted that this study does not permit a full assessment of reproductive and developmental toxicity considering the very limited numbers of parameters assessed in this study compared to OECD test guidelines.

In a 2-generation study (Anonymous, 1978a), male and female Wistar rats received MeS in the diet at 2500 and 5000 ppm (equivalent to 125 and 250 mg/kg bw MeS) for 60 days prior to mating, then throughout the study (until weaning of the offspring). Each generation of rats was mated twice. Examinations performed were very limited: mating performance, number of pups, stillbirths, live birth, postnatal mortality, gross abnormalities, physical and behavioural abnormalities. An increase of unsuccessful mating for the first generation (21.7% vs 8% in control with no mating) and a decrease in reproduction index for both generations [number of weaned 21 days/number of liveborn * 100] (76.2% vs 82.7% in the first generation and 76.9% vs 89.8% in the second generation) were reported at the highest dose. A decrease of litter size was noted at all doses. Higher number of deaths between birth and day 5 was also observed at 500 mg/kg bw/day. Only results of statistical analysis for total born, live born and total weaned/female were reported but not statistical significant effect was found. No gross abnormalities were observed in young born. All young surviving to weaning appeared normal in respect to body growth, appearance and behavior. Considering the low quality of the study and results, no adequate NOAEL can be set. It should be noted that this study does not permit a full assessment of reproductive and developmental toxicity considering the very limited numbers of parameters assessed in this study compared to OECD test guidelines.

In a further 2 generation study (Anonymous,1978b), male and female mice were exposed to MeS at 2500 and 5000 ppm (equivalent to 375 and 750 mg/kg bw MeS) from 30 days prior to mating until weaning of offspring. Examinations performed were very limited: mating performance, number of pups, stillbirths, live birth, postnatal mortality, gross abnormalities, physical and behavioural abnormalities. The only effect reported is a “slightly smaller litter size” in test groups at birthhowever no statistical analysis was performed. Thus the relevance of this effect cannot be adequately assessed. Considering the low quality of the study and results, no adequate NOAEL can be set. It should be noted that this study does not permit a full assessment of reproductive and developmental toxicity considering the very limited numbers of parameters assessed in this study.

A last study was summarized in the CIR (2003) review. Groups of 24 to 27 SD rats were fed a diet containing 4000 or 6000 ppm of MeS and calcium carbonate for 60 days prior to mating (FDA, 1966). The dams were fed the test diets until the neonates were weaned at day 20 or 21, and the procedure was repeated with a second mating. No abnormalities were observed in the offspring of test animals. Neonate survival at weaning was greater in the test group than in the control group. This study cannot be adequately assessed due to the very limited level of details available.

Effects on developmental toxicity

Description of key information

The lowest NOAEL for developmental toxicity can be set at < 60 mg/kg bw/day (but > 20 mg/kg bw/day) based on skeletal variations.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

unpublished report cited by FDA and RAC (ANSES has access to the original studies cited in the FDA report, and considers this study as the Key study with a Klimisch reliability of 1)

Guideline:

other: ICh guideline

Principles of method if other than guideline:

- Principle of test: Study for effects on Embryo-fetal development
- Short description of test conditions: pregnant New Zealand White rabbits were exposed to methyl salicylate by subcutaneous administration from gestation day 6 to gestation day 18 at 3 different dose levels.

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Route of administration:

subcutaneous

Vehicle:

corn oil

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 mL/kg
- Concentration (if solution): 30, 100 or 300 mg/mL

Duration of treatment / exposure:

from gestation day 6 to gestation day 18

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

18-20 females/group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the administration and once daily during other periods

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 3, 6-19, 23, 26, and 29 of gestation

FOOD CONSUMPTION : Yes
- Food consumption was recorded on days 1, 3, 6-19, 23, 26 and 29

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29

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: Yes
- Number of late resorptions: Yes

Blood sampling:

blood was drawn from 4 dams per group at 4 hours post-dosing on GD6 and 18 for toxicokinetic analysis.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data

Details on maternal toxic effects:

One dam at the highest dose had an abortion on gestation day 18, with a complete late resorption. Slight not significant depression in body weight gain (without impact on body weight) was observed throughout the administration period at 300 mg/kg/day. A NOAEL of 100 mg/kg/d is set for maternal toxicity based on these effects. There was no treatment related effect on the numbers of corporea lutea, implants or live fetuses, dead embryo / foetus indices or body weight of live fetuses. A significant, but not dose-related, decrease in the pre-implant loss index (66.7%) as compared with the control group was observed in the 30 mg/kg group. Since implantation occurs before treatment to methyl salicylate, this effect is not considered related to treatment. There was a significant difference in sex ratio, with a larger number of male fetuses (↑ 44.4%) in the 300 mg/kg/d as compared with the control group. However, sex determination occurs genetically on day 6 of gestation or before.
The degree of elevation of the plasma salicylic acid concentration was nearly dependent on increases in the dose ratio. Plasma concentration of salicylic acid was scarcely affected by repeated dosing.

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

There was no placental anomaly, no external, visceral or skeletal anomalies related to methyl salicylate treatment. The NOAEL for development toxicity is 300 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect pbserved

Abnormalities:

no effects observed

Developmental effects observed:

no

Executive summary:

In this study (summarized in FDA 2006 and RAC opinion 2019), pregnant New Zealand White rabbits (18-20/group) were exposed to methyl salicylate by subcutaneous administration from gestation day 6 to gestation day 18 at the doses of 0, 30, 100 or 300 mg/kg/day. The highest dose was selected based on a preliminary study showing mortality from the dose of 500 mgkg/day and pre-implant loss index from 250 mg/kg/day. In the main study, one dam at the highest dose had an abortion on gestation day 18, with a complete late resorption. Slight not significant depression in body weight gain (without impact on body weight) was observed throughout the administration period at 300 mg/kg/day. A NOAEL of 100 mg/kg/d is set for maternal toxicity based on these effects. There was no treatment related effect on the numbers of corporea lutea, implants or live fetuses, dead embryo / foetus indices or body weight of live fetuses. A significant, but not dose-related, decrease in the pre-implant loss index (66.7%) as compared with the control group was observed in the 30 mg/kg group. Since implantation occurs before treatment to methyl salicylate, this effect is not considered related to treatment. There was a significant difference in sex ratio, with a larger number of male fetuses (↑ 44.4%) in the 300 mg/kg/d as compared with the control group. However, sex determination occurs genetically on day 6 of gestation or before. There was no placental anomaly, no external, visceral or skeletal anomalies related to methyl salicylate treatment. The NOAEL for development toxicity is 300 mg/kg/day. The degree of elevation of the plasma salicylic acid concentration was nearly dependent on increases in the dose ratio. Plasma concentration of salicylic acid was scarcely affected by repeated dosing.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

unpublished report cited by FDA and RAC (ANSES has access to the original studies cited in the FDA report, and considers this study as the Key study with a Klimisch reliability of 1)

Guideline:

other: ICh guideline

Principles of method if other than guideline:

- Principle of test: Study for effects on Embryo-fetal development
- Short description of test conditions: female rats were treated with methyl salicylate by subcutaneous administration from gestation day 6 to gestation day 17 at 3 different dose levels (definitive segment II study).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crj:CD(SD)IGS

Route of administration:

subcutaneous

Vehicle:

corn oil

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 mL/kg
- Concentration (if solution): 50, 100 or 200 mg/mL

Duration of treatment / exposure:

from gestation day 6 to gestation day 17

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 females/group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the administration and once daily during other periods

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 3, 6-20 of gestation

FOOD CONSUMPTION : Yes
- Food consumption was recorded on days 1, 3, 6-20

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20

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: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes (control and 200 mg/kg/d groups only)
- Skeletal examinations: Yes
- Head examinations: No data

Details on maternal toxic effects:

No mortality or clinical signs occurred in the treated groups. Statistically significant depression of body weight (< 5%), body weight gain (≥ 10%) and food consumption was reported in dams at 200 mg/kg/day. A transient statistically significant decreased body weight gain was also observed at 100 mg/kg/day without any significant impact on the body weight. A NOAEL of 100 mg/kg/day is set for maternal toxicity based on the decreased body weight. There was no effect of the treatment on the number of corporea lutea, implants, live and dead fetuses, sex ratio or placental anomalies.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Details on embryotoxic / teratogenic effects:

Lower body weight of live fetuses (- 22%) was observed at 200 mg/kg/day. In the highest dose group, there was an increase of external anomalies (3.21% versus 0.36% in the control), characterized principally by craniorachischisis (8 foetuses in 3 litters equivalent to 2.86% versus 0% in the control group) and gastroschisis (1 foetus). Even not clearly indicated in the report, these anomalies should be considered as malformations (Devtox.org). Although not statistically significant, it should be noted that, based on historical control data for development and reproductive toxicity studies using the Crl:CD®BR rat compiled by MARTA (1993), craniorachischisis and gastroschisis are rarely observed in rats (both with average fetal incidence of 0.01%). In this context, the incidence of 2.86% reported in the FDA (2006c) study is clearly above the MARTA historical controls in rat experiments (1993). In addition, these effects are considered by the authors as related to methyl salicylate treatment because they are consistent with the results of the preliminary study and with available data reported in the literature with methyl salicylate and salicylic acid. Visceral anomalies (ventricular septal defect (considered as malformation according to Devtox.org) in one foetus, dilatation of the ureter (unilateral) in 2 foetuses and thymic remnant in the neck in 8 foetuses) were also increased at 200 mg/kg/day (7.75% versus 3.52% in the control) but not statistically significant. A statistically significant increase of skeletal variations was also observed at the highest dose (75.19% versus 10.61% in the control group), with short and full supernumerary ribs, splitting of the thoracic and lumbar vertebral bodies, 7 lumbar vertebrae and incomplete ossification of the thoracic centrum. In addition, there was a delay of ossification of the vertebrae, sternebra, metacarpus, metatarsus and phalanges. In conclusion, methyl salicylate is considered teratogenic in rats. The NOAEL for developmental toxicity is 100 mg/kg/day based on external malformations, visceral anomalies, decreased fetal body weight, skeletal variations and delayed ossification.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

Developmental effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Executive summary:

In this study (summarized in FDA 2006 and RAC opinion 2019), pregnant Crj:CD(SD)IGS rats (20/group) were exposed to methyl salicylate by subcutaneous administration from gestation day 6 to gestation day 17 at the doses of 0, 50, 100 or 200 mg/kg/day. The highest dose was selected based on a preliminary study showing mortality at the dose of 400 mgkg/day and decreased maternal body weight gain and embryolethality at the dose of 300 mg/kg/day. In the main study, no mortality or clinical signs occurred in the treated groups. Statistically significant depression of body weight (< 5%), body weight gain (≥ 10%) and food consumption was reported in dams at 200 mg/kg/day. A transient statistically significant decreased body weight gain was also observed at 100 mg/kg/day without any significant impact on the body weight. A NOAEL of 100 mg/kg/day is set for maternal toxicity based on the decreased body weight. There was no effect of the treatment on the number of corporea lutea, implants, live and dead fetuses, sex ratio or placental anomalies. Lower body weight of live fetuses (- 22%) was observed at 200 mg/kg/day. In the highest dose group, there was an increase of external anomalies (3.21% versus 0.36% in the control), characterized principally by craniorachischisis (8 foetuses in 3 litters equivalent to 2.86% versus 0% in the control group) and gastroschisis (1 foetus). Even not clearly indicated in the report, these anomalies should be considered as malformations (Devtox.org). Although not statistically significant, it should be noted that, based on historical control data for development and reproductive toxicity studies using the Crl:CD®BR rat compiled by MARTA (1993), craniorachischisis and gastroschisis are rarely observed in rats (both with average fetal incidence of 0.01%). In this context, the incidence of 2.86% reported in the FDA (2006c) study is clearly above the MARTA historical controls in rat experiments (1993). In addition, these effects are considered by the authors as related to methyl salicylate treatment because they are consistent with the results of the preliminary study and with available data reported in the literature with methyl salicylate and salicylic acid. Visceral anomalies (ventricular septal defect (considered as malformation according to Devtox.org) in one foetus, dilatation of the ureter (unilateral) in 2 foetuses and thymic remnant in the neck in 8 foetuses) were also increased at 200 mg/kg/day (7.75% versus 3.52% in the control) but not statistically significant. A statistically significant increase of skeletal variations was also observed at the highest dose (75.19% versus 10.61% in the control group), with short and full supernumerary ribs, splitting of the thoracic and lumbar vertebral bodies, 7 lumbar vertebrae and incomplete ossification of the thoracic centrum. In addition, there was a delay of ossification of the vertebrae, sternebra, metacarpus, metatarsus and phalanges. In conclusion, methyl salicylate is considered teratogenic in rats. The NOAEL for developmental toxicity is 100 mg/kg/day based on external malformations, visceral anomalies, decreased fetal body weight, skeletal variations and delayed ossification.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

unpublished report cited by FDA and RAC (ANSES has access to the original studies cited in the FDA report, and considers this study as the Key study with a Klimisch reliability of 1)

Guideline:

other: ICH guideline

Principles of method if other than guideline:

- Principle of test: Study for effects on pre- and postnatal development, including maternal function
- Short description of test conditions: female rats were treated with methyl salicylate by subcutaneous administration from gestation day 6 to gestation day 21 at 3 different dose levels (definitive segment III study).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crj:CD(SD)IGS

Route of administration:

subcutaneous

Vehicle:

corn oil

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 mL/kg

Duration of treatment / exposure:

from gestation day 6 to gestation day 21

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 females/group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the administration and once daily during other periods

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: on days 3, 6, 9, 12, 15, 18, and 20 of gestation, and on days 0, 4, 7, 10, 14, 17, and 21 of lactation period

FOOD CONSUMPTION : Yes
- Food consumption was recorded on days 1, 3, 6, 9, 12, 15, 18, and 20 of gestation, and on days 1, 4, 7, 10, 14, 17, and 20 of lactation period

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 22 after delivery

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Fetal examinations:

At birth: number of litter, number of stillborns, number of live newborns,still birth index. Live offsprings were weighed, sexed and examined for external anomalies.
During lactation and after weaning: function test (righting reflex and ipsilateral flexor (PND5), visual pacing reflex (PND6), Preyer's reflex (PND28)), postnatal differenciation test (pinna detachment (PND4), incisor eruption (PND10), gait and eyelid separation (PND15), descensus testis (PND31), cleavage of the balanopreputial gland (PND42), and vaginal opening (PND42).
Birth to PND22: testing of the F1 litter included daily examination for clinical signs and mortality. Body weight of pups was recorded in day 0, 7, 14 and 21 after birth.
Organ weight: 1 offspring of each sex from a dam was euthanasia on PND22. The following organs were isolated and weighed: heart, lungs, liver, kidneys, adrenals, brain, spleen, thymus, and testes and ovaries.
Skeletal examination: on PND22, 2 offsprings of each sex from a dam were euthanized. Their organs and tissues were observed macroscopically. Skeletal anomalies and variations were assessed after staining with alizarin red S.
PND22 (weaning) until mating: offsprings were observed daily for clinical signs and mortality. Body weight and food consumption were recorded once a week. Neurobehavioral evaluation included: rotarod performance (5 weeks of age), water maze (6 weeks of age), and open field (8 weeks of ages) were performed.
F1 Reproductive capacity: At 12 to 13 weeks, the F1 capacity to reproduce was evaluated. Duration of matinf required for copulation, copulation index, and male and female fertility indices were assessed.

Details on maternal toxic effects:

Two dams at 200 mg/kg/day died on gestation day 23. These death were considered to have been induced by aggravation of their general condition attributable to methyl salicylate. There was a significantly lower mean body weight (-3.7% on GD12 and -4.6% on GD20) and body weight gain (between -4.08% on GD9 and -15.7% on GD20) during gestation at 200 mg/kg/day. The food consumption was significantly decreased on day 9 of gestation (-10.2%) and during lactation (-42.9% on day 1 and -21.9% on day 21) at this same dose. A significant prolongation of gestational days was observed in the 60 mg/kg/day group (with no dose-response relationship and within background data of the institution).

Regarding reproductive ability of the offspring, there was no significant difference in the copulation indices (95%, 85%, 95%, 94%), fertility indices (100%, 100%, 94.74%, 80%) and in the numbers of days required for copulation. A significant lower body weight was observed in F1 dams on gestation day 13 at the highest dose. At necropsy of the males after mating, excessive elongation of the maxillary was observed in 1 male and corectopia and dyscoria in another male at 200 mg/kg/day. In the necropsy of the females on gestation day 13, corectopia and dyscoria were observed in 1 female at 200 mg/kg/day. There was an increase of pre-implantation losses (7.18 versus 1.99) at the highest dose but not statistically significant.

Dose descriptor:

NOAEL

Effect level:

ca. 60 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

In male offspring, a significant decrease in the birth index (-6%) and a lower body weight (-9.2%) were observed in live newborn in the 200 mg/kg/day group. A trend toward a decrease in the number of litter (215 litters at 200 mg/kg bw/day versus 270 in the control group) and live newborns and a trend toward an increase in the stillbirth index (7 stillborns at 200 mg/kg bw/day versus 2 in the control group) were also observed in the 200 mg/kg/day group. These effects were considered attributable to methyl salicylate administration. No abnormality was reported in the external examination of the live newborn but craniorachischisis was noted in 4 stillborns (among 6 stillborns reported in 4 females; there is no indication in how many litters craniorachischisis occurred) in the 200 mg/kg/day group. A trend toward a decrease in the viability index (92.79%) on day 4 was observed at the highest dose compared to control (98.13%) but was within the range of the background data (91.32-99.28%). Excessive elongation of the maxillary incisors (1 female; 2 males), corectopia and dycoria (1 male; 1 female) were reported at 200 mg/kg/day. A significant lower mean body weight with decreased food consumption was noted during lactation and maturation in the 200 mg/kg/day group. A significant decrease in the differentiation indices of incisor eruption in both sexes (64% in males and 56% in females versus 100 % in controls of both sexes on PND12), eyelid separation in the females (85% versus 100% on PND15 in controls) and cleavage of the balanopreputial gland in the males (67% versus 100% in controls on PND46) were reported at the highest dose. In the males at weaning, a significant decrease in the absolute and relative weights of the liver and kidneys, in the absolute weights of the brain, adrenals and testes and a significant increase in the relative weights of the brain and lungs was observed at 200 mg/kg/day. In females, a significant decrease in the absolute weights of the brain, heart, lungs, liver, kidneys, adrenals and ovaries and a significant increase in the relative weight of the brain were noted at 200 mg/kg/day.
Skeletal anomalies, especially fusion of the cervical vertebra and misshapen sternebra, were significantly increased at 200 mg/kg/day (32.26% versus 3.90% in the control). Skeletal variations slightly increased at 60 mg/kg bw/day (cervical ribs, accessory sternebra, incomplete ossification of thoracic and caudal vertebrae) and was significantly increased at 200 mg/kg/day (93.55% versus 25.97% in the control), with full supernumerary ribs, accessory sternebra, lumbarization, 7 lumbar vertebrae and incomplete ossification of the cervical, thoracic and lumbar centrum. No historical control data was presented. Considering that these effects were also identified in other prenatal developmental toxicity studies, it could not be ruled out that the variations occurring at 60 mg/kg/day are treatment-related. A significant decrease of the number of rearing occurred in F1 female offspring at 200 mg/kg/day (8.1 versus 12.6) but was within laboratory control (6.0-8.7).
Regarding reproductive ability of the offspring, there was no significant difference in the copulation indices (95%, 85%, 95%, 94%), fertility indices (100%, 100%, 94.74%, 80%) and in the numbers of days required for copulation. A significant lower body weight was observed in F1 dams on gestation day 13 at the highest dose. At necropsy of the males after mating, excessive elongation of the maxillary was observed in 1 male and corectopia and dyscoria in another male at 200 mg/kg/day. In the necropsy of the females on gestation day 13, corectopia and dyscoria were observed in 1 female at 200 mg/kg/day. There was an increase of pre-implantation losses (7.18 versus 1.99) at the highest dose but not statistically significant.

Dose descriptor:

NOAEL

Effect level:

>= 0 - < 60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

other: decrease in differentiation indices in the highest tested group

Abnormalities:

effects observed, treatment-related

Localisation:

other: Craniorachischisis, maxillary incisors, vertebrae, ribs

Developmental effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Executive summary:

In this study (summarized in FDA 2006 and RAC opinion 2019), 20 pregnant female rats per group were exposed subcutaneously to methyl salicylate at 0, 20, 60 or 200 mg/kg/day from gestation day 6 to lactation day 21. Dams were sacrificed on day 22 after delivery. The highest tested dose was selected based on a preliminary study showing mortality in almost all dams at 500 mg/kg/day, no live delivery at 300 mg/kg/day and slight effect on birth index and body weight at 80 and 200 mg/kg/day.
Two dams at 200 mg/kg/day died on gestation day 23. These death were considered to have been induced by aggravation of their general condition attributable to methyl salicylate. There was a significantly lower mean body weight (-3.7% on GD12 and -4.6% on GD20) and body weight gain (between -4.08% on GD9 and -15.7% on GD20) during gestation at 200 mg/kg/day. The food consumption was significantly decreased on day 9 of gestation (-10.2%) and during lactation (-42.9% on day 1 and -21.9% on day 21) at this same dose. A significant prolongation of gestational days was observed in the 60 mg/kg/day group (with no dose-response relationship and within background data of the institution).
In male offspring, a significant decrease in the birth index (-6%) and a lower body weight (-9.2%) were observed in live newborn in the 200 mg/kg/day group. A trend toward a decrease in the number of litter (215 litters at 200 mg/kg bw/day versus 270 in the control group) and live newborns and a trend toward an increase in the stillbirth index (7 stillborns at 200 mg/kg bw/day versus 2 in the control group) were also observed in the 200 mg/kg/day group. These effects were considered attributable to methyl salicylate administration. No abnormality was reported in the external examination of the live newborn but craniorachischisis was noted in 4 stillborns (among 6 stillborns reported in 4 females; there is no indication in how many litters craniorachischisis occurred) in the 200 mg/kg/day group. A trend toward a decrease in the viability index (92.79%) on day 4 was observed at the highest dose compared to control (98.13%) but was within the range of the background data (91.32-99.28%). Excessive elongation of the maxillary incisors (1 female; 2 males), corectopia and dycoria (1 male; 1 female) were reported at 200 mg/kg/day. A significant lower mean body weight with decreased food consumption was noted during lactation and maturation in the 200 mg/kg/day group. A significant decrease in the differentiation indices of incisor eruption in both sexes (64% in males and 56% in females versus 100 % in controls of both sexes on PND12), eyelid separation in the females (85% versus 100% on PND15 in controls) and cleavage of the balanopreputial gland in the males (67% versus 100% in controls on PND46) were reported at the highest dose. In the males at weaning, a significant decrease in the absolute and relative weights of the liver and kidneys, in the absolute weights of the brain, adrenals and testes and a significant increase in the relative weights of the brain and lungs was observed at 200 mg/kg/day. In females, a significant decrease in the absolute weights of the brain, heart, lungs, liver, kidneys, adrenals and ovaries and a significant increase in the relative weight of the brain were noted at 200 mg/kg/day.
Skeletal anomalies, especially fusion of the cervical vertebra and misshapen sternebra, were significantly increased at 200 mg/kg/day (32.26% versus 3.90% in the control). Skeletal variations slightly increased at 60 mg/kg bw/day (cervical ribs, accessory sternebra, incomplete ossification of thoracic and caudal vertebrae) and was significantly increased at 200 mg/kg/day (93.55% versus 25.97% in the control), with full supernumerary ribs, accessory sternebra, lumbarization, 7 lumbar vertebrae and incomplete ossification of the cervical, thoracic and lumbar centrum. No historical control data was presented. Considering that these effects were also identified in other prenatal developmental toxicity studies, it could not be ruled out that the variations occurring at 60 mg/kg/day are treatment-related. A significant decrease of the number of rearing occurred in F1 female offspring at 200 mg/kg/day (8.1 versus 12.6) but was within laboratory control (6.0-8.7).
Regarding reproductive ability of the offspring, there was no significant difference in the copulation indices (95%, 85%, 95%, 94%), fertility indices (100%, 100%, 94.74%, 80%) and in the numbers of days required for copulation. A significant lower body weight was observed in F1 dams on gestation day 13 at the highest dose. At necropsy of the males after mating, excessive elongation of the maxillary was observed in 1 male and corectopia and dyscoria in another male at 200 mg/kg/day. In the necropsy of the females on gestation day 13, corectopia and dyscoria were observed in 1 female at 200 mg/kg/day. There was an increase of pre-implantation losses (7.18 versus 1.99) at the highest dose but not statistically significant. The NOAEL for maternal toxicity is set at 60 mg/kg/day based on decreased body weight and food consumption. According to the authors, the NOAEL for developmental toxicity is < 60 mg/kg/day based on the slight and non-statistically significant increased incidence of skeletal variations at 60 mg/kg bw/day. An increase in lethality and skeletal anomalies / variations and a decrease in differentiation indices and body weight were noted in the highest tested group.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Summary of the data as reported in the CLH dossier and RAC opinion:

In a first study (summarized in FDA, 2006b), pregnant New Zealand White rabbits (18-20/group) were exposed to methyl salicylate by subcutaneous administration from gestation day 6 to gestation day 18 at the doses of 0, 30, 100 or 300 mg/kg/day. The highest dose was selected based on a preliminary study showing mortality from the dose of 500 mgkg/day and pre-implant loss index from 250 mg/kg/day. In the main study, one dam at the highest dose had an abortion on gestation day 18, with a complete late resorption. Slight not significant depression in body weight gain (without impact on body weight) was observed throughout the administration period at 300 mg/kg/day. A NOAEL of 100 mg/kg/d is set for maternal toxicity based on these effects. There was no treatment related effect on the numbers of corporea lutea, implants or live fetuses, dead embryo / foetus indices or body weight of live fetuses. A significant, but not dose-related, decrease in the pre-implant loss index (66.7%) as compared with the control group was observed in the 30 mg/kg group. Since implantation occurs before treatment to methyl salicylate, this effect is not considered related to treatment. There was a significant difference in sex ratio, with a larger number of male fetuses (↑ 44.4%) in the 300 mg/kg/d as compared with the control group. However, sex determination occurs genetically on day 6 of gestation or before. There was no placental anomaly, no external, visceral or skeletal anomalies related to methyl salicylate treatment. The NOAEL for development toxicity is 300 mg/kg/day. The degree of elevation of the plasma salicylic acid concentration was nearly dependent on increases in the dose ratio. Plasma concentration of salicylic acid was scarcely affected by repeated dosing.

In a second study (summarized in FDA, 2006c), pregnant Crj:CD(SD)IGS rats (20/group) were exposed to methyl salicylate by subcutaneous administration from gestation day 6 to gestation day 17 at the doses of 0, 50, 100 or 200 mg/kg/day. The highest dose was selected based on a preliminary study showing mortality at the dose of 400 mgkg/day and decreased maternal body weight gain and embryolethality at the dose of 300 mg/kg/day. In the main study, no mortality or clinical signs occurred in the treated groups. Statistically significant depression of body weight (< 5%), body weight gain (≥ 10%) and food consumption was reported in dams at 200 mg/kg/day. A transient statistically significant decreased body weight gain was also observed at 100 mg/kg/day without any significant impact on the body weight. A NOAEL of 100 mg/kg/day is set for maternal toxicity based on the decreased body weight. There was no effect of the treatment on the number of corporea lutea, implants, live and dead fetuses, sex ratio or placental anomalies. Lower body weight of live fetuses (- 22%) was observed at 200 mg/kg/day. In the highest dose group, there was an increase of external anomalies (3.21% versus 0.36% in the control), characterized principally by craniorachischisis (8 foetuses in 3 litters equivalent to 2.86% versus 0% in the control group) and gastroschisis (1 foetus). Even not clearly indicated in the report, these anomalies should be considered as malformations (Devtox.org). Although not statistically significant, it should be noted that, based on historical control data for development and reproductive toxicity studies using the Crl:CD®BR rat compiled by MARTA (1993), craniorachischisis and gastroschisis are rarely observed in rats (both with average fetal incidence of 0.01%). In this context, the incidence of 2.86% reported in the FDA (2006c) study is clearly above the MARTA historical controls in rat experiments (1993). In addition, these effects are considered by the authors as related to methyl salicylate treatment because they are consistent with the results of the preliminary study and with available data reported in the literature with methyl salicylate and salicylic acid. Visceral anomalies (ventricular septal defect (considered as malformation according to Devtox.org) in one foetus, dilatation of the ureter (unilateral) in 2 foetuses and thymic remnant in the neck in 8 foetuses) were also increased at 200 mg/kg/day (7.75% versus 3.52% in the control) but not statistically significant. A statistically significant increase of skeletal variations was also observed at the highest dose (75.19% versus 10.61% in the control group), with short and full supernumerary ribs, splitting of the thoracic and lumbar vertebral bodies, 7 lumbar vertebrae and incomplete ossification of the thoracic centrum. In addition, there was a delay of ossification of the vertebrae, sternebra, metacarpus, metatarsus and phalanges. In conclusion, methyl salicylate is considered teratogenic in rats. The NOAEL for developmental toxicity is 100 mg/kg/day based on external malformations, visceral anomalies, decreased fetal body weight, skeletal variations and delayed ossification.

 

In a third study (summarized in FDA (2006d)), 20 pregnant female rats per group were exposed subcutaneously to methyl salicylate at 0, 20, 60 or 200 mg/kg/day from gestation day 6 to lactation day 21. Dams were sacrificed on day 22 after delivery. The highest tested dose was selected based on a preliminary study showing mortality in almost all dams at 500 mg/kg/day, no live delivery at 300 mg/kg/day and slight effect on birth index and body weight at 80 and 200 mg/kg/day. Two dams at 200 mg/kg/day died on gestation day 23. These death were considered to have been induced by aggravation of their general condition attributable to methyl salicylate. There was a significantly lower mean body weight (-3.7% on GD12 and -4.6% on GD20) and body weight gain (between -4.08% on GD9 and -15.7% on GD20) during gestation at 200 mg/kg/day. The food consumption was significantly decreased on day 9 of gestation (-10.2%) and during lactation (-42.9% on day 1 and -21.9% on day 21) at this same dose. A significant prolongation of gestational days was observed in the 60 mg/kg/day group (with no dose-response relationship and within background data of the institution).
In male offspring, a significant decrease in the birth index (-6%) and a lower body weight (-9.2%) were observed in live newborn in the 200 mg/kg/day group. A trend toward a decrease in the number of litter (215 litters at 200 mg/kg bw/day versus 270 in the control group) and live newborns and a trend toward an increase in the stillbirth index (7 stillborns at 200 mg/kg bw/day versus 2 in the control group) were also observed in the 200 mg/kg/day group. These effects were considered attributable to methyl salicylate administration. No abnormality was reported in the external examination of the live newborn but craniorachischisis was noted in 4 stillborns (among 6 stillborns reported in 4 females; there is no indication in how many litters craniorachischisis occurred) in the 200 mg/kg/day group. A trend toward a decrease in the viability index (92.79%) on day 4 was observed at the highest dose compared to control (98.13%) but was within the range of the background data (91.32-99.28%). Excessive elongation of the maxillary incisors (1 female; 2 males), corectopia and dycoria (1 male; 1 female) were reported at 200 mg/kg/day. A significant lower mean body weight with decreased food consumption was noted during lactation and maturation in the 200 mg/kg/day group. A significant decrease in the differentiation indices of incisor eruption in both sexes (64% in males and 56% in females versus 100 % in controls of both sexes on PND12), eyelid separation in the females (85% versus 100% on PND15 in controls) and cleavage of the balanopreputial gland in the males (67% versus 100% in controls on PND46) were reported at the highest dose. In the males at weaning, a significant decrease in the absolute and relative weights of the liver and kidneys, in the absolute weights of the brain, adrenals and testes and a significant increase in the relative weights of the brain and lungs was observed at 200 mg/kg/day. In females, a significant decrease in the absolute weights of the brain, heart, lungs, liver, kidneys, adrenals and ovaries and a significant increase in the relative weight of the brain were noted at 200 mg/kg/day. Skeletal anomalies, especially fusion of the cervical vertebra and misshapen sternebra, were significantly increased at 200 mg/kg/day (32.26% versus 3.90% in the control). Skeletal variations slightly increased at 60 mg/kg bw/day (cervical ribs, accessory sternebra, incomplete ossification of thoracic and caudal vertebrae) and was significantly increased at 200 mg/kg/day (93.55% versus 25.97% in the control), with full supernumerary ribs, accessory sternebra, lumbarization, 7 lumbar vertebrae and incomplete ossification of the cervical, thoracic and lumbar centrum. No historical control data was presented. Considering that these effects were also identified in other prenatal developmental toxicity studies, it could not be ruled out that the variations occurring at 60 mg/kg/day are treatment-related. A significant decrease of the number of rearing occurred in F1 female offspring at 200 mg/kg/day (8.1 versus 12.6) but was within laboratory control (6.0-8.7).
Regarding reproductive ability of the offspring, there was no significant difference in the copulation indices (95%, 85%, 95%, 94%), fertility indices (100%, 100%, 94.74%, 80%) and in the numbers of days required for copulation. A significant lower body weight was observed in F1 dams on gestation day 13 at the highest dose. At necropsy of the males after mating, excessive elongation of the maxillary was observed in 1 male and corectopia and dyscoria in another male at 200 mg/kg/day. In the necropsy of the females on gestation day 13, corectopia and dyscoria were observed in 1 female at 200 mg/kg/day. There was an increase of pre-implantation losses (7.18 versus 1.99) at the highest dose but not statistically significant. The NOAEL for maternal toxicity is set at 60 mg/kg/day based on decreased body weight and food consumption. According to the authors, the NOAEL for developmental toxicity is < 60 mg/kg/day based on the slight and non-statistically significant increased incidence of skeletal variations at 60 mg/kg bw/day. An increase in lethality and skeletal anomalies / variations and a decrease in differentiation indices and body weight were noted in the highest tested group.

 

Developmental effects are also reported in fertility studies:
Additional information on developmental toxicity can be obtained from the fertility studies.

In a study of fertility and early embryonic development to implantation in rats (FDA, 2006a), there was no significant effect on early development of embryos (numbers of implants or live embryos, pre-implant low index or dead embryo index) at doses up to 300 mg/kg bw/day of methyl salicylate by subcutaneous route.

In a continuous breeding protocol study (NTP, 1984a), including task 2 and 4, in mice, there was no adverse effects reported in the number of pups per litter, percentages of live pups or pup weight and at necropsy of F1 at doses up to 100 mg/kg bw/day of methyl salicylate in the diet. There was also no treatment-related effect on parental survival, body weight and food consumption.

In a second continuous breeding protocol study (NTP, 1984b), including task 2 and 3, in mice exposed to methyl salicylate in the diet, reduced pup viability was reported at the high dose of 500 mg/kg bw/day with decrease in the mean number of litter (-8%), in the average of live pups per litter (-31%) and the proportion of pups born alive (-6%). At 250 mg/kg bw/day, a reduction in pup weight (about -4%) was reported in females. The NOAEL for development was set at 100 mg/kg bw/day based on the decrease of pup body weight. No treatment-related effect on parental mortality, clinical signs and body weight was reported.

In a 3-generation study performed by Collins et al. (1971) in rats exposed to methyl salicylate in the diet, adverse effects were reported on offspring such as decreases in average litter size (2nd generation: mating 1: 10.8, 10.2, 10.3, 8.4, 6.2 at 0, 500, 1500, 3000 and 5000 ppm respectively; mating 2: 11.9, 10.2, 10.5, 9.4, 6.6 at 0, 500, 1500, 3000 and 5000 ppm respectively), number of liveborn progeny per female, viability (liveborn) (2nd generation: mating 1: 10.8, 10.2, 10.2, 8.2, 5.6 at 0, 500, 1500, 3000 and 5000 ppm respectively; mating 2: 11.8, 10.2, 10.5, 9.1, 6.3 at 0, 500, 1500, 3000 and 5000 ppm respectively), survival (survivors on day 4) (2nd generation: mating 1: 9.4, 9.0, 9.5, 6.2, 4.3 at 0, 500, 1500, 3000 and 5000 ppm respectively; mating 2: 11.1, 9.4, 10.3, 8.2, 4.7 at 0, 500, 1500, 3000 and 5000 ppm respectively) and weaning survival (2nd generation: mating 1: 8.8, 8.4, 9.4, 6.0, 3.9 at 0, 500, 1500, 3000 and 5000 ppm respectively; mating 2: 10.5, 8.6, 9.9, 8.0, 3.7 at 0, 500, 1500, 3000 and 5000 ppm respectively). These effects are only statistically significant in the 2nd generation, with a dose-related decrease starting from 1500 ppm (75 mg/kg bw/day). Decreases in weight at the weaning (up to – 21%) appeared consistently from 3000 ppm (equivalent to 150 mg/kg bw/day). There was no external abnormality or histopathological effect on the liver and kidney of offspring of the 3rd generation at weaning. The NOAEL for development was 75 mg/kg bw/day based on pup mortality and decreased weight. It should be noted that this study does not permit a full assessment of reproductive and developmental toxicity considering the limited numbers of parameters assessed in this study. In addition, there was no information on general parental toxicity reported in the publication.

In a 2-generation study (Anonymous, 1978a) performed in rats exposed to methyl salicylate in the diet, a decrease of litter size was noted at both doses tested (10.61, 9.00, 9.74 at 0, 2500 and 5000 ppm, respectively – equivalent to 125 and 250 mg/kg bw/day). Higher number of deaths between birth and day 5 was also observed at 500 mg/kg bw/day (alive at 5 days: 8.46 versus 10.04 in control group). Only results of statistical analysis for total born, live born and total weaned/female were reported but not statistical significant effect was found. No gross abnormalities were observed in young born. All young surviving to weaning appeared normal in respect to body growth, appearance and behavior. Considering the low quality of the study and results, no adequate NOAEL can be set. It should be noted that this study does not permit a full assessment of reproductive and developmental toxicity considering the very limited numbers of parameters assessed in this study. In addition, there was no information on general parental toxicity reported in the report.

In a further 2 generation study (Anonymous,1978b) performed in mice exposed to methyl salicylate in the diet, the only effect reported is a “slightly smaller litter size” in test groups (2500 and 5000 ppm equivalent to 375 and 750 mg/kg bw/day) at birth (11.53, 10.95, 10.35 at 0, 2500 and 5000 ppm, respectively), however no statistical analysis was performed. Thus the relevance of this effect cannot be adequately assessed. Considering the low quality of the study and results, no adequate NOAEL can be set. It should be noted that this study does not permit a full assessment of reproductive and developmental toxicity considering the very limited numbers of parameters assessed in this study. In addition, there was no information on general parental toxicity reported in the report.

In a one-generation study summarized in the CIR (2003) review, no abnormalities were observed in the offspring of test animals exposed to 4000 ppm and 6000 ppm (equivalent to 200 and 300 mg/kg bw/day) of methyl salicylate. Neonate survival at weaning was greater in the test groups than in the control group. This study cannot be adequately assessed due to the very limited level of details available.

Studies of lower quality are also available:
Pregnant rats received dermal application of undiluted MeS or diluted in a petroleum based grease. Undiluted MeS was initially applied at 2000 mg/kg bw/day from GD6 but due to severe toxicity (dermal irritation and 25% mortality), the dose was reduced to 1000 mg/kg bw/day from GD10 to GD15. At this dose, a 100% resorption was reported, but there was no information on maternal toxicity after reduction of the dose (Infurna et al., 1990 – only abstract available).

MeS was administered topically at 3500 and 5250 mg/kg bw to pregnant LVG hamsters on day 7 and teratogenic results were compared with those obtained following oral treatment at 1750 mg/kg bw. After dermal exposure for 2 hours, the skin was thoroughly washed with running water. Blood samples were obtained at regular intervals to monitor salicylate. Most embryos were recovered at GD9, few survived to the age of 12 days. Both treatments produced neural tube defects, especially in the area of the developing brain. Percentage of neural tube defect was 72% at 1750 mg/kg bw/day after oral exposure versus 11% in control. After dermal exposure, the percentage of neural tube defect was 0% in control, 6% at 3500 mg/kg bw/day and 53% at 5250 mg/kg bw/day. Analysis of serum showed that salicylate levels reached a peak of 125 mg/100 ml at about 2 hours after oral administration and 50 mg/100 ml at 5-6h after dermal application. Comparison of maternal and fetal salicylate levels in older fetuses showed that salicylate was reaching the foetus in some fraction of the concentration found in the mother (Overman & White, 1983).

Other studies were available and described in different reviews (RIFM, 2007; Lapczynski et al., 2007 and CIR, 2003).
Female rats received 0.05 or 0.1 mL MeS by intraperitoneal route on days 10 and 11 of pregnancy. The young were obtained on GD21 or postnatally at 1, 6, 12 or 24 days of age. They were counted, weighted and examined for viability and external malformations. Kidneys were removed, weighted and examined. At 0.1 mL, females gained less weight, had fewer and smaller offspring and more resorptions and malformed young than in the control group. Fetal kidneys weighted significantly less than those of the controls and lengthening of the renal papilla was inhibited by MeS, suggesting that MeS can induce renal growth retardation. Additionally, there was a significantly higher frequency of kidneys with absent papillae. Retarded renal development recovered on PND6, but persistent hydronephrosis (11/138 kidneys) was still observed at weaning. It is not clear from the publication if these effects are only observed at the highest tested dose or at both doses (Woo et al., 1972).

Daston et al (1988) performed several experiments where MeS was given by intraperitoneal route to pregnant rats from 200 to 450 mg/kg bw/day, on different gestation days and for different durations. Malformations, reduction of fetal weight and some increase in the incidence of resorption were reported. On this basis, a further study was performed to study postnatal renal function of offspring. Pregnant rats were exposed to 200-300 mg/kg bw/day MeS on GD 11-12. Increased mortality during the first 2 days after birth was noted from 250 mg/kg bw/day. Increase in kidney/body weight ratio was observed on day 15 but not by 4 weeks of age.

In a last study performed in rats by intraperitoneal route at 200 and 400 mg/kg bw/day on GD9 and 10, decreased fetal weight, reduction of fetal body weight index and malformations were reported at both tested doses in the presence of maternal toxicity (Kavlock et al., 1982).

As conclusions, developmental effects, mainly characterized by lethality, external malformations, visceral and skeletal anomalies and effects on differentiation indices, were reported in developmental and reproductive studies with methyl salicylate. The lowest developmental NOAEL are < 60 mg/kg bw/day in rats exposed subcutaneously from gestation day 6 to lactation day 21 (FDA, 2006b) and 75 mg/kg bw/day in a 3-generation study in rats by oral route (Collins et al. 1971).

Justification for classification or non-classification

Fertility:

Not classified for effects on reproduction (fertility) according to CLP criteria.

Multigeneration studies on the effects of the substance on fertility in rats and mice indicate that it does not adversely affect fertility at any dose at any generation.

 

Development:

Not classified for effects on reproduction (development) according to CLP criteria.

Based on a weight of evidence approach assessing data from animal studies and human data on acetylsalicylic acid concluding that ASA, and therefore MeS, have no developmental toxicity effects in humans when doses applied are representative of actual Human exposure (medical treatment at least, at doses higher than the rat NOAELs).(see Discussion section and Read-across justification in Chapter 13).