[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data are available on the reproductive toxicity of ATMP-xNH4, therefore good quality data from the category member, ATMP-acid are read-across.

The key reproductive study is a multigenerational study for the category member, ATMP-H (100% active acid), conducted prior to the adoption of OECD test guidelines and pre-GLP. Male and female Long-Evans rats were administered ATMP 60 days prior to mating (F0) and continuously thereafter (F1, F2, F3) in the diet at fixed concentrations of 0, 300, 1000 and 3000 ppm for three consecutive generations. The NOAEL for general toxicity and reproductive toxicity was at least the highest dose tested of 3000 ppm. The concentration of the test substance and mean weekly food intake values were used to determine the approximate doses received by the animals. The dose of 3000 ppm was approximately equal to a dose of 275 mg active acid/kg bw/day in males and 310 mg active acid/kg bw/ day in females (Biodynamics Inc., 1979a).

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29.10.1976 to 15.08.1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Three generation reproduction toxicity study with the following restrictions: no assessment of estrus cycle, sperm parameters, sexual milestones, no analytical confirmation of exposure levels.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Altamont, New York.
- Age at study initiation: (P) 6-7 weeks
- Weight at study initiation: (P) males approx. 370 g, females approx. 240 g at mating
- Fasting period before study: No data
- Housing: Individually (except during mating and lactation) in elevated stainless steel wire mesh cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days.


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: From: 12.11.1976 To: 15.08.1978

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Laboratory Chow (standard laboratory diet)
- Storage temperature of food: No data

Details on mating procedure:

- M/F ratio per cage: 1/2 (See table 1)
- Length of cohabitation: Overnight for up to 15 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- Remated (for F1b/F2b/F3b generation) following a 14 d rest period.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged: individually in elevated stainless steel wire mesh cages.
- Any other deviations from standard protocol: None apparent.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet samples were taken from control and treated groups weekly. Samples were stored frozen and sent to the sponsor at regular intervals throughout the study. No further details.

Duration of treatment / exposure:

From 60 days prior to first mating of P generation then continuous over 3 generations . Duration of test in total was approximately 21 months.

Frequency of treatment:

Daily

Details on study schedule:

- F1 and F2 parental animals not mated until after a growth period (unspecified duration) following selection from the F1b and F2b litters.
- Selection of parents from F1 and F2 generation when pups were 7 days post weaning.
- Age at mating of the mated animals in the study: Not clear, but there was a 14 day rest period between matings.

Dose / conc.:

300 ppm (nominal)

Remarks:

nominal in diet
See table 3 for conversion to mg active acid/kg bw/day

Dose / conc.:

1 000 ppm (nominal)

Remarks:

nominal in diet
See table 3 for conversion to mg active acid/kg bw/day

Dose / conc.:

3 000 ppm (nominal)

Remarks:

nominal in diet
See table 3 for conversion to mg active acid/kg bw/day

No. of animals per sex per dose:

12 male and 24 females (see Table 1)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random

Positive control:

None

Parental animals: Observations and examinations:

Examination conducted on F0, F1, F2 AND F3 (all adult generations)

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Gross signs twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during growth and rest periods of all animals. As well as pregnant females (F0, F1b, F2b) on GD 0, 6, 15 and 20 and lactating females (F0, F1) on LD 0, 4, 14 and 21.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly for males and non-pregnant females.
- 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


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: F0 parents after weaning of F1b litter.

Oestrous cyclicity (parental animals):

Not investigated.

Sperm parameters (parental animals):

Not investigated.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, to 10 pups/sex/litter as nearly as possible; excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioral abnormalities. See Tables 4, 5 and 6 for pup survival data.


GROSS EXAMINATION OF DEAD PUPS:
yes, sex determined and stomach checked for presence of milk. Cause of death was not determined.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after completion of pup selection for the F0 and F1 generations, and after weaning of last litters for the F2 generation.
- Maternal animals: All surviving animals after completion of pup selection for the F0 and F1 generations, and after weaning of last litters for the F2 generation. Also non-pregnant dams from first mating.
- Dead and moribund animals examined as death occurred.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Dams uterine contents examined for the presence of implantation sites and/or scars.


HISTOPATHOLOGY / ORGAN WEIGHTS: None scheduled for adults.Only grossly abnormal tissues were examined.

Postmortem examinations (offspring):

SACRIFICE
- The F1a/F2a/F3a offspring not selected as parental animals were sacrificed at 21 days of age.
- F1b and F2b progeny (non-parental): sacrificed after pup selection for next generation.
- F3b sacrificed at weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 2 were prepared for microscopic examination from 10 pups/sex/group of the F3b generation. In addition any grossly abnormal tissues were examined.

Statistics:

Offspring body weights, off-spring numbers (LD 0 LD 4): F-test and Student's T-test.
Offspring survival, litter deaths, litters weaned, mortality, mating rates, pregnancy rates, fertility rates: Chi square.
Body weights, body weight change, food intake: Dunnett's test.

Reproductive indices:

mating indices (%), pregnancy rates (%) and fertility (%). No details given.

Offspring viability indices:

No details given.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Physical observations comparable between all groups.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One mid dose male was sacrificed in a moribund state during mating for F1b litter ("severely tilted head"). One high dose female was sacrificed post-weaning of F1b litter with "extremely distended abdomen". There was no dose response and the deaths were considered sporadic, therefore not related to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect in either sex on mean body weight or body weight gain during growth or rest periods. There were no effects on maternal body weight or body weight change during gestation or lactation periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See Table 3.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The ophthalmoscopic examination revealed four rats (one control female, two mid dose males and one high dose male) with ocular abnormalities. However, these were not considered to be treatment-related.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Gestation length, mean number of live and dead pups at birth and percentage of live pups at birth were comparable between groups. A high number of dead pups within a single mid dose litter lead to a significant decrease in the survival index at birth in the mid dose group for F1b.

Key result

Dose descriptor:

NOAEL

Remarks:

F0 / P0

Effect level:

>= 3 000 ppm

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 (parental animals): Severely tilted heads in one control male and one high dose female. Red and swollen ears on the tagged ear were noted in F1 and F2 adults.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

F1: One mid dose male died during mating. One high dose female died during post-weaning interval, prior to sacrifice. One control male and one high dose female sacrificed with "severely tilted heads" (male sacrificed week 4, female in rest period between matings). There was no dose response and the deaths were considered sporadic, therefore not related to treatment. Physical observations comparable between all groups.
F2: One low dose female died during week 3 of the growth period. One mid dose male died during week 8 of the growth period. One mid dose female died on GD 21 for the F3b litter. One high dose male died during the mating interval to produce the second litter. There was no dose response, and the deaths were considered sporadic, therefore not related to treatment. Physical observations comparable between all groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect in either sex on mean body weight or body weight gain during growth or rest periods. There were no effects on maternal body weight or body weight change during gestation or lactation periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See Table 3.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no adverse findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Gestation length, mean number of live and dead pups at birth and percentage of live pups at birth were comparable between groups. A high number of dead pups within a single mid dose litter lead to a significant decrease in the survival index at birth in the mid dose group for F1b. The F2 generation high dose group had a significantly higher survival index at birth for the second litters. No adverse effect was concluded. There was no effect on mating indices (%), pregnancy rates (%) or fertility (%).

Key result

Dose descriptor:

NOAEL

Remarks:

F1 / P1

Effect level:

>= 3 000 ppm

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

F2 / P2

Effect level:

>= 3 000 ppm

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

None reported.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Significant decrease (P<0.01) in survival index at birth in the second mid dose litter (F1 for F2b). This was not considered indicative of a treatment-related effect by the study authors since comparable changes this was not replicated in other phases of the study, nor was any dose/response relationship present. Survival at birth was significantly increased (P<0.01) for the high dose F3b litter. Some statistically significant differences were apparent in postnatal survival indices between control and treated groups; however these were not considered adverse by the authors since no trend was present. Comment: these differences generally reflected a statistically significant enhancement in survival relative to the controls (9 instances), while decreased survival was relatively infrequent (2 instances). The percentages of litters with offspring deaths and litters weaned were comparable between control and treated groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Comparable between all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No adverse findings.

Histopathological findings:

no effects observed

Description (incidence and severity):

No adverse findings.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

F1

Generation:

F1

Effect level:

>= 3 000 ppm

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

None reported.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Significant decrease (P<0.01) in survival index at birth in the second mid dose litter (F1 for F2b). This was not considered indicative of a treatment-related effect by the study authors since comparable changes this was not replicated in other phases of the study, nor was any dose/response relationship present. Survival at birth was significantly increased (P<0.01) for the high dose F3b litter. Some statistically significant differences were apparent in postnatal survival indices between control and treated groups, however these were not considered adverse by the authors since no trend was present. Comment: these differences generally reflected a statistically significant enhancement in survival relative to the controls (9 instances), while decreased survival was relatively infrequent (2 instances). The percentages of litters with offspring deaths and litters weaned were comparable between control and treated groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Comparable between all groups.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Scattered red foci present in lung from some F2b offspring from the mid and high dose groups (not present in controls and low dose group) considered unrelated to treatment by authors (since not present in other generations). All other necropsy observations similar for control and treated litters. Evaluation of selected tissues from 10 control weanlings and 10 high dose weanlings from the F3b generation revealed no abnormalities. Changes present in lung consistent with minimal to mild interstitial pneumonia, microscopic appearance of the gonads unremarkable and consistent with sexually immature rats.

Histopathological findings:

no effects observed

Description (incidence and severity):

No adverse findings.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

F2

Generation:

F2

Effect level:

>= 3 000 ppm

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

F3

Generation:

other: F3

Effect level:

>= 3 000 ppm

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Critical effects observed:

no

Reproductive effects observed:

no

Table 3 Test substance intake based on food intake (weekly mean data) measurements .

	Males (mg/kg bw/day)	Males (mg/kg bw/day)	Males (mg/kg bw/day)	Females (mg/kg bw/day)	Females (mg/kg bw/day)	Females  (mg/kg bw/day)
Group (ppm)	II (300)	III (1000)	IV (3000)	II (300)	III (1000)	IV (3000)
F0 growth	33.4	111.6	342.3	37.3	117.1	362.5
F0 rest	18.4	60.7	182.9	24.3	75.7	247.2
F1 growth	26.3	89.6	270.2	28.1	98.4	290.2
F1 rest	14.4	41.7	141.0	20.1	67.3	201.1
F2 growth	29.6	95.4	296.6	34.3	112.6	337.8
F2 rest	17.6	58.3	171.9	25.0	81.4	243.9

Table 4 Summary of offspring survival for the F1 generation.

Group (ppm)	Mean gestation length	% pups born alive	Mean no weaned/litter	Postnatal survival (%)	Postnatal survival (%)	Postnatal survival (%)	% litters with death (days 0 -21)^c	% litters weaned^c	Sex ratio (M/F)
				0 -4^a	4 -14^b	14 -21
F0 to F1a
I (0)	22.5	98.8	9.1	92.0	94.3	100	57.1	95.2	0.87
II (300)	22.4	98.2	8.6	97.3*	90.5	100	65	100	0.87
III (1000)	22.5	98.1	7.6*	96.2	85.4**	100	47.6	95.2	0.88
IV (3000)	22.3	98.1	8.7	99.2**	91.4	99	52.2	100	1.24
F0 to F1b
I (0)	22.1	93.0	8.9	87.2	96.0	99.3	61.1	88.9	1.07
II (300)	22.1	96.0	8.7	91.7	91.3	99.4	83.3	100	1.12
III (1000)	22.1	97.2	9.3	94.8*	93.7	100	56.3	100	0.84
IV (3000)	22.1	95.8	9.2	97.6**	96.5	99.5	28.6	100	0.96

Significantly different from control *p≤0.05; **p≤0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.

Table 5 Summary of offspring survival for the F2 generation.

Group (ppm)	Mean gestation length	% pups born alive	Mean no weaned/litter	Postnatal survival (%)	Postnatal survival (%)	Postnatal survival (%)	% litters with death (days 0 -21)^c	% litters weaned^c	Sex ratio (M/F)
				0 -4^a	4 -14^b	14 -21
F1 to F2a
I (0)	22.2	99.5	9.2	96.2	93.5	100	33.3	94.4	1.09
II (300)	22.3	100	9.5	96.8	99.5**	100	35.0	100	1.18
III (1000)	22.1	99.6	9.4	95.4	99.5**	98.6	40.9	100	1.06
IV (3000)	22.3	97.4	9.1	97.3	100**	100	30.4	100	0.92
F1 to F2b
I (0)	22.4	99.3	8.8	78.5	100	100	35.7	85.7	0.89
II (300)	22.1	96.1	8.8	93.6**	92.5	98.1	61.1	100	1.11
III (1000)	22.1	92.5**	8.2	92.4**	88.5**	58.8	93.8	93.8	0.95
IV (3000)	22.5	99.1	9.0	96.6**	97.8	98.9	50.0	100	0.94

Significantly different from control *p≤0.05; **p≤0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.

Table 6 Summary of offspring survival for the F3 generation.

Group (ppm)	Mean gestation length	% pups born alive	Mean no weaned/litter	Postnatal survival (%)	Postnatal survival (%)	Postnatal survival (%)	% litters with death (days 0 -21)^c	% litters weaned^c	Sex ratio (M/F)
				0 -4^a	4 -14^b	14 -21
F2 to F3a
I (0)	22.3	97.9	7.8	89.5	94.3	99.4	54.5	95.5	0.91
II (300)	22.4	91.3	6.4	87.2	89.5	100	75.0	100	1.13
III (1000)	22.2	96.2	7.7	85.9	89	100	57.1	87.7	1.09
IV (3000)	22.2	98.6	8.8	96.7**	91.9	99.4	66.7	94.7	1.11
F2 to F3b
I (0)	22.2	92.9	9.1	89.5	97.4	98.6	50.0	88.9	1.00
II (300)	22.4	96.1	8.7	92.4	98.5	99.2	43.8	93.8	1.06
III (1000)	22.1	92.7	8.3	93.5	97.3	100	38.5	100	0.83
IV (3000)	22.2	98.5**	9.2	89.9	98.7	100	52.9	94.1	1.45

Significantly different from control *p≤0.05; **p≤0.01

aComparison between days for postnatal offspring survival are calculated using Day 4 pre-cull data.

bComparison between days for postnatal offspring survival are calculated using Day 4 post-cull data.

cOnly those pups found alive at Day 0 of lactation are used in calculations.

Conclusions:

In a reasonably well conducted three generation reproductive toxicity study, conducted before the adoption of OECD test guidelines and GLP, the general and reproductive toxicity NOAEL for ATMP-H (100% active acid) was greater than the highest dose tested, 3000 ppm in the diet, in rats (approximately equal to a dose of 275 mg active acid/kg bw/day in males and 310 mg active acid/kg bw/day for females).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

275 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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 data are available on the reproductive toxicity of ATMP-xNH4, therefore good quality data from the category member, ATMP-H are read-across. See attachment to IUCLID Section 13 for justification of read-across.

The key reproductive study is a multigenerational study for the category member, ATMP-H (100% active acid), and is judged to be reliable with restrictions. Male and female Long-Evans rats were administered ATMP 60 days prior to mating (F0) and continuously thereafter (F1, F2, F3) in the diet at fixed concentrations of 0, 300, 1000 and 3000 ppm for three consecutive generations. The litters from F0, F1 and F2 matings were raised to maturity and also mated. Offspring from the first litter of each generation (F1a, F2a, F3a) were taken for necropsy on lactation Day 21. The parents were remated following a 14-day rest period and the offspring randomly selected at seven days post-weaning to continue as the F1b and F2b generation parents. Remaining F1b and F2b animals, as well as the F3a and F3b generation, were taken for necropsy. A gross internal examination was conducted on these animals. Randomly selected offspring from the F3a litters (10 pups/sex/group) were necropsied and selected tissues examined microscopically. Evaluations of adult mortality, mating, pregnancy, fertility, body weight data, food consumption data (growth and rest periods), litter survival, offspring viability at parturition, offspring weight and sex, and necropsy of adults and offspring, did not indicate any treatment related adverse effects. The NOAEL for general toxicity and reproductive toxicity was greater than the highest dose tested, 3000 ppm. The concentration of the test substance and mean weekly food intake values were used to determine the approximate doses received by the animals. 3000 ppm was approximately equal to a dose of 275 mg active acid/kg bw/day in males and 310 mg active acid/kg bw/ day in females (Biodynamics Inc., 1979a).

Effects on developmental toxicity

Description of key information

No data are available on the developmental toxicity of ATMP-xNH4, therefore good quality data from studies on rats and mice are read-across from the category member, ATMP-H. See attachment to IUCLID Section 13 for justification of read-across..

 

In the key developmental toxicity study, conducted prior to the adoption of OECD test guidelines and preGLP, ATMP-H (aqueous solution containing 22.4% w/w active acid) was administered by oral gavage to pregnant Charles River SD rats (24/dose) on gestation days 6-15. The doses tested were 100, 500 or 1000 mg active acid/kg bw/day (measured as the active acid). The maternal NOAEL was concluded to be 500 mg active acid/kg bw/day based on lower body weight gain during the treatment period in females at 1000 mg/kg bw/day when compared to the control animals; the change was considered to be treatment-related by the study authors; the NOAEL for fetotoxicity and teratogenicity was concluded to be ≥1000 mg active acid/kg bw/day respectively. There were no other treatment-related effects observed in the study (BioDynamics Inc., 1979b).

In the key developmental toxicity study, conducted prior to the adoption of OECD test guidelines and pre-GLP, ATMP-H (aqueous solution containing 20% w/w active acid) was administered by oral gavage to pregnant CD-1 mice (35 mated females/dose) on gestation days 6-15. The doses tested were 100, 500 and 1000 mg/kg bw/day. The doses are assumed to be as active acid but this is not clearly specified in the study report. If the doses were in terms of solution as provided, the equivalent active acid values would be 20, 100, 200 mg active acid/kg bw/day (based on 20% active acid). The NOAEL for maternal toxicity, foetal toxicity and teratogenicity was concluded to be at least 1000 mg/kg bw/day (Biodynamics Inc., 1980).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data. Dates of treatment were 27.12.1978 to 19.01.1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The study is a read across from ATMP (CAS 6419-19-8).

Guideline:

other: FDA "Guidelines for reproductive studies for evaluation of drugs for human use", segment II (teratological study)

Deviations:

not specified

Remarks:

Treatment on GD 6 - 15; no record of gravid uterine weight; number corpora lutea not recorded; no analytical confirmation of exposure levels.

Principles of method if other than guideline:

Study was used to assess the teratogenic and/or embryotoxic potential of the test substance.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

72 d at mating
Weight: approx. 240 - 250 Number: 24 / dose Supplier: Charles River, Wilmington, Mass. Individually housed, food (Purina Certified Rodent Chow 5002) and water available ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

ADMINISTRATION / EXPOSURE GD 6-15 Test substance mixed with water, administered at 10 mg/kg/d. Volume adjusted based on most recent body weight data. Prepared daily.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Overnight
- Further matings after two unsuccessful attempts: No data
- Verification of same strain and source of both sexes: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.

Duration of treatment / exposure:

GD 6 - 15

Frequency of treatment:

daily

Duration of test:

16 days

Dose / conc.:

100 mg/kg bw/day

Remarks:

measured as the active acid

Dose / conc.:

500 mg/kg bw/day

Remarks:

measured as the active acid

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

measured as the active acid

No. of animals per sex per dose:

24 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: 21 d

Maternal examinations:

Clinical observations performed and frequency: - gross signs, twice daily - detailed physical examination of GD 0, 6, 10, 15, 20 and 21 (pre-necropsy) Parent: - bwt: GD 0, 6 -15, 21
- Organs examined at necropsy Dams sacrificed on GD21 by lethal exposure to diethyl ether.
Parent: - complete post-mortem examination - uterus: live / dead foetuses, late / early resorptions, implantation sites - ovaries: corporea lutea per ovary

Fetal examinations:

Fetal: - crown-rump distance - sex (anogenital distance) - external malformations - approx. 50% of foetuses subject to gross dissection and visceral examination followed by processing / staining (Alizarin red) for skeletal abnormalities / variations - remainder subject to Wilson serial sectioning for neural / visceral defects (10X or 20X magnification) after fixing in Bouin's solution

Statistics:

Chi squared or F-test and Student's T-test. T-tests modified using Cochran's approximation when variances differed significantly. Live foetuses, resorptions, implantations and corpora lutea compared using one-tailed T-test.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs in any dose groups were found.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female in the 100 mg/kg bw/day dose group was concluded to be in moribund condition and was sacrificed on GD6 (first day of treatment).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were found in maternal body weight gain between groups. The body weight gain GD6-15: 50/49/50/44 - 12% (non-significant) reduction in body weight gain at 1000 mg/kg bw/d on GD6-15. Individual body weight gain for dam 822 (high dose) on GD6-15 = 22 g; mean gain for controls = 50 g; mean gain for high dose group = 44 g. Body weight gain for this dam on GD 0-6 (preceding treatment) and on GD 15-21 (post-treatment) was similar or greater than mean bdoy weight gain for control and high dose group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were observed in any of the dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not specified

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A statistical significant 7.5 % increase in implantation efficiency at 100 mg/kg bw/day was observed. However, this was considered unrelated to the test substance by authors as the implantation (14.5/14.7/14.0/13.7) and implantation efficiency (84.3%/91.8%/84.0%/81.2%) was comparable.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Mean number of resorptions were 0.2/0.8/0.5/0.8 which were all within historical control range data, There was an increase although not statistical significant increase in dams with 2 or more resorptions in treated groups (0%/22.7%/8.3%/20.8%), However, this was also within historical control range and therefore, considered to not be test substance treatment-related effects.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were observed in any of the dose groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The pregnancy rate was comparable between all dose groups (100% in control, mid and high dose groups, 95.6% at 100 mg/kg bw/d).

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of corpora lutea were 17.2/16.0/16.7/16.8. The 100 mg/kg bw/day dose group had a 7% decrease in corpora lutea. However, this was considered by the authors to be unrelated to the treatment

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

act. ingr.

Remarks:

active acid

Basis for effect level:

other: Effects on body weigh gain.

Abnormalities:

no effects observed

Fetal body weight changes:

not specified

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex: males/litter 6.9/7.0/6.6/6.0; females 7.4/6.9/7.0/6.9 (no significant effect). Sex ratio (m:f): 92.4%/102.0%/94.6%/87.3% (no significant effect)

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The body weight for males were 5.54/5.43/5.71/5.49 and for females 5.25/5.17/5.34/5.16 which did not indicate any significant effect.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 mg/kg bw/day dose group, one female had 6 foetuses (from a total litter of 16) with a syndrome of defects that included: flexed forepaws, shortened and thickened torso, abdominal distension and exaggerated forward flexure of the head (see maternal body weight, above). No other animals, including the other animals in the 1000 mg/kg bw/day dose group, had any external malformations and thereby, this effect were considered to be incidental and not related to treatment of the test substance.

Description (incidence and severity):

Total of foetuses examined: 177/158/169/159 - per foetus: 4.0%/1.9%/3.0%/1.3% (no significant effect) - per litter: 20.8%/13.6%/20.8%/8.7% (no significant effect). In the control dose group, angulated ribs, cervical rib and wavy rib were observed. In the 100 mg/kg bw/day dose group, angulated rib, cervical rib as well as angulated and wavy rib were observed. In the 500 mg/kg bw/day dose group. cervical rib, angulated and wavy rib as well as 7 lumbar vertebra were observed. In the 1000 mg/kg bw/day dose group, 5 lumbar vertebra and fused sternebrae were observed. There was no significant variation in the ossification: fetuses 80.2%/83.5%/79.3%/84.3%; litters 95.8%/100.0%/100.0%/100.0%.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Total foetuses examined: 162/147/156/150 - per fetus: 4.3%/8.2%/4.5%/4.0% (no significant effect) - per litter: 16.7%/40.9%/29.2%/20.8% (no significant effect). In the control dose group, distended renal pelvis, renal pelvis, ureter and bladder malformations were observed. In the 100 mg/kg bw/day dose group, a similar incidence in the control dose group as well as a fold in retina. In the 500 mg/kg bw/day dose group, a similar incidence as in the control dose group as well as ectopic kidney. In the 1000 mg/kg bw/day dose group, a similar incidence as in the control dose group as well as fold in retina, anophthalmia, malrotation of heart occurring in two foetuses from the same litter which had an altered maternal body weight and observed external malformations.
The incidence of visceral malformations per fetus were: 0.6%/1.3%/0.6%/1.9% (no significant effect) - per litter: 4.2%/9.1%/4.2%/12.% (no significant effect). In the control dose group, distended ureter was observed. In the 100 mg/kg bw/day, a distended ureter +/- renal pelvis occurred. In the 500 mg/kg bw/day dose group, a similar incidence occurred as in the control dose group. In the 1000 mg/kg bw/day, a similar incidence occurred as in the control dose group as well as malpositioned testis were observed.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Crown-rump length: males 4.2/4.2/4.3 (P<0.01)/4.2; females 4.1/4.1/4.2 (P<0.01)/4.1. There was an increase at 500 mg/kg bw/day, however, this was not considered be of biological insignificance by authors.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No teratogenic effects observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

active acid

Sex:

male/female

Basis for effect level:

other: No fetotoxic effects observed.

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In a well-documented pre-GLP teratology study (FDA segment II teratological study; reliability score 2) ATMP-H (aqueous solution containing 22.4% w/w active acid) was not embryotoxic or teratogenic when administered to rats at 100 or 500 mg active acid/kg bw/day (measured as the active acid) by gavage on GD6-15. At 1000 mg/kg bw/d, six fetuses from a single litter showed common multiple malformations in presence of a 50% decrease in individual maternal body weight gain (possibly indicative of concurrent maternal toxicity); all other high dose fetuses were normal. The clear absence of any comparable effect in other high dose litters and lack of dose-response indicates that 1000 mg active acid/kg bwt/d was the no-effect level for embryotoxicity and fetotoxicity. The maternal NOAEL was 500 mg active acid/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data. Treatment days were 09.04.1980 to 13.05.1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Guideline:

other: FDA "Guidelines for reproductive studies for evaluation of drugs for human use", segment II (teratological study)

Deviations:

not specified

Remarks:

No analytical evaluation of exposure levels.

Principles of method if other than guideline:

The study was designed to evaluate the embryotoxic and/or teratogenic potential of the test substance. Dosing was on gestation days 6-15, no measurement of gravid uterine weights and copora lutea were not counted.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc.
- Age at study initiation: Females 57 days (males stated to be sexually mature)
- Weight at study initiation: Females on gestation Day 0 were approximately 26 g.
- Fasting period before study: No data
- Housing: Individual in elevated stainless steel cages.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: One month


ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data. Monitored twice daily.
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: Day 6 of gestation: 09.04.1980 - 04.05.1980 To: Day 15 of gestation: 18.04.1980 - 13.05.1980.

Route of administration:

oral: gavage

Vehicle:

other: Not clear, stated to be distilled water and corn oil in different parts of the report.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of test substance were dissolved in distilled water and administered at a constant volume of 10 ml/kg bw/day. Dosing solutions were prepared fresh daily.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Overnight
- Verification of same strain and source of both sexes: No data
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.

Duration of treatment / exposure:

GD 6 - 15

Frequency of treatment:

daily

Duration of test:

18 days

Dose / conc.:

100 mg/kg bw/day

Remarks:

The dose is assumed to be as active acid but this is not clearly specified in the study report. If the dose was in terms of solution as provided, the equivalent active acid value would be 20 mg active acid/kg bw/day (based on 20% active acid).

Dose / conc.:

500 mg/kg bw/day

Remarks:

The dose is assumed to be as active acid but this is not clearly specified in the study report. If the dose was in terms of solution as provided, the equivalent active acid value would be 100 mg active acid/kg bw/day (based on 20% active acid).

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

The dose is assumed to be as active acid but this is not clearly specified in the study report. If the dose was in terms of solution as provided, the equivalent active acid value would be 200 mg active acid/kg bw/day (based on 20% active acid).

No. of animals per sex per dose:

35 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes for mortality and gross signs of toxicological effects (no further details).
- Time schedule: Twice daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Gestation days 0, 6, 9, 12, 15 and 18.


BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 6, 9, 12, 15 and 18. Calculated body weight change for days 0-6, 6-15 and 15-18.


FOOD CONSUMPTION: No


WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18 (all surviving dams) and Day 18 post-mating in all surviving non-pregnant females.
- Organs examined: Complete gross pathology examination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes, and weighed, measured and sex determined.
- Other: Live and dead fetuses. Internal sex determination of fetuses.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: No data

Statistics:

Maternal body weight and reproduction data: Bartlett's test followed by one-way ANOVA (equal variance) followed by Dunnett's test or Kruskal-Wallis test (unequal variance) and summed rank test (Dunn).  Pregnancy and fetal parameters: Chi square analysis followed by Fisher Exact test with Bonferroni correction. Armitage test for linear trend.

Indices:

No data

Historical control data:

No data

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse clinical effects.

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related deaths (only deaths that occurred as a result of dosing errors and one death of a control animal on gestation day 11).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects on body weights or body weight gains.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related adverse findings during the macroscopic examination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No effects on implantations.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No effects on resorptions.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

No effects on numbers of live and dead fetuses.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

No effects on pregnancy rates.

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There were no treatment-related deaths (only deaths that occurred as a result of dosing errors and one death of a control animal on gestation day 11), no adverse clinical effects, no effects on body weights or body weight gains, and no effects on reproductive parameters (pregnancy rates, numbers of live and dead fetuses, implantations and resorptions). There were no treatment-related adverse findings during the macroscopic examination.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks:

Assumed to be as active acid but not clearly specified in the study report. If the dose was in terms of solution as provided, the equivalent active acid value would be 200 mg active acid/kg bw/day (based on 20% active acid).

Basis for effect level:

other: No adverse maternal toxicity effects observed.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No effects on mean fetal body weights.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No effects on fetal sex distribution.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The incidence of fetal external malformations were comparable between control and treated groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

During the fetal skeletal evaluations, the incidence of fetuses with at least one ossification variation was slightly higher than the concurrent control in the mid and high dose groups. However, incidences for these groups were within the range of historical values for the laboratory and strain of mouse. The type and incidence of ossification variations during the skeletal evaluations were similar to the controls. However there was a slight increase in the incidence of fetuses with rudimentary structures observed in the mid and high dose groups.
During the skeletal evaluations the incidence of malformations was low in the low (no malformations observed) and high dose groups. The incidence of skeletal malformations in the mid-dose group was significantly increased. However, this increase was attributed to a high number of malformed fetuses from a single mid-dose litter. Six fetuses from this litter had skeletal malformations that included misshapen tibia and fibula, angulated ribs and defective sternebrae. Since these effects were not observed in the highest and lowest dose groups they were not considered to be related to treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

The incidence of fetal soft tissue malformations were comparable between control and treated groups. No malformations were noted in the treated groups during the gross evisceration examinations.

Other effects:

no effects observed

Description (incidence and severity):

No effects on crown-rump length.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks:

Assumed to be as active acid but not clearly specified in the study report. If the dose was in terms of solution as provided, the equivalent active acid value would be 200 mg active acid/kg bw/day (based on 20% active acid).

Sex:

male/female

Basis for effect level:

other: No adverse teratogenic effects observed.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks:

Assumed to be as active acid but not clearly specified in the study report. If the dose was in terms of solution as provided, the equivalent active acid value would be 200 mg active acid/kg bw/day (based on 20% active acid).

Sex:

male/female

Basis for effect level:

other: No adverse fetotoxic effects were observed.

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1 Summary of Malformations found during the fetal skeletal examination.

Group (mg/kg bw/day)	Malformation	Fetuses		Litter
		No. with malf./total examined	%	No. with malf fetuses/total examined	%
0	None	0/138		0/24
100	Cervical vertebral defect	1/156	0.6	1/28	3.6
500	Tibia misshapened - alone	1/203	0.5	1/34	2.9
	- with misshapened fibula and angulated ribs	2/203	1.0	1/34	2.9
	- with misshapened fibula, angulated ribs and sternebrae defects	1/203	0.5	1/34	2.9
	- with angulated ribs	1/203	0.5	1/34	2.9
	Angulated ribs and scrambled sternebrae	1/203	0.5	1/34	2.9
	Cervical rib	1/203	0.5	1/34	2.9
	5 lumbar vertebrae	2/203	1.0	1/34	2.9
	Total	9/203	4.4*	3/34	8.8
1000	Scrambled sternebrae	1/183	0.5	1/32	3.1
	Vertebral defects	1/183	0.5	1/32	3.1

*Difference from the control group statistically significant p<0.05 (Fisher Exact test).

Conclusions:

In a well-conducted teratogenicity study (FDA segment II: teratological study; reliability score 2) conducted prior to the adoption of OECD test guidelines and GLP, it was concluded that ATMP-H (aqueous solution containing 20% w/w active acid) was not embryotoxic or teratogenic when administered to mice at 100, 500 or 1000 mg/kg bw/d ay by gavage on GD6-15. The NOAEL for maternal toxicity, foetal toxicity and teratogenicity was greater than 1000 mg/kg bw/day. The doses are assumed to be as active acid but this is not clearly specified in the study report. If the doses were in terms of solution as provided, the equivalent active acid values would be 20, 100, 200 mg active acid/kg bw/day (based on 20% active acid).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 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:

no study available

Additional information

No data are available on the developmental toxicity of ATMP-xNH4, therefore good quality data from studies on rats and mice are read-across from the category member, ATMP-H. See attachment to IUCLID Section 13 for justification of read-across.

In the key developmental toxicity study, ATMP-H (aqueous solution containing 22.4% w/w active acid) was administered by oral gavage to pregnant Charles River SD rats (24/dose) on gestation days 6-15. The doses tested were 100, 500 or 1000 mg active acid/kg bw/day (measured as the active acid). Control animals received the vehicle (water) only. Dams were sacrificed on gestation day 21 and recovered foetuses evaluated for external, soft-tissue and skeletal malformations. Maternal mortality, pregnancy rate, body weight gain, uterine implantation data, foetal size, sex data, ossification variation data and teratological evaluations were evaluated. High dose females gained less weight than the controls during the dosing period. In the high dose group, six foetuses from a single litter had common multiple malformations that included flexed forepaws, shortened and thickened torso, abdominal distension and exaggerated flexure of the head. Soft tissue examination revealed two of these foetuses had a malformation defect of the heart. The findings were observed in one litter only to a dam showing signs of toxicity in the form of reduced body weight gain of 50%. Therefore, the malformations were considered as secondary to maternal toxicity. No other foetuses in the high dose group had any effects.

The maternal NOAEL was concluded to be 500 mg active acid/kg bw/day based on lower body weight gain during the treatment period in females at 1000 mg/kg bw/day when compared to the control animals; the change was considered to be treatment-related by the study authors; the NOAEL for fetotoxicity and teratogenicity was concluded to be at least 1000 mg active acid/kg bw/day respectively. There were no other treatment-related effects observed in the study (BioDynamics Inc., 1979b).

In the key developmental toxicity study, ATMP-H (aqueous solution containing 20% w/w active acid) was administered by oral gavage to pregnant CD-1 mice (35 mated females/dose) on gestation days 6 -15. The doses tested were 100, 500 and 1000 mg/kg bw/day whereas the control group received the vehicle only. The doses are assumed to be as active acid but this is not clearly specified in the study report. If the doses were in terms of solution as provided, the equivalent active acid values would be 20, 100, 200 mg active acid/kg bw/day (based on 20% active acid). Parameters for evaluation included mortality, body weight, clinical signs and uterine implantation data, ossification variation data and teratological evaluation. The occurring deaths in the treated animals depended on dosing errors and one death of a control animal occurred on gestation day 11. No adverse clinical effects, effects on body weights or body weight gains, effects during the macroscopic examination or on reproductive parameters (pregnancy rates, numbers of live and dead fetuses, implantations and resorptions) occurred. In the fetuses, the fetal sex distribution and anogenital distance were unaffected by the treatment. The NOAEL for maternal toxicity, fetal toxicity and teratogenicity was concluded to be at least 1000 mg/kg bw/day (BioDynamics Inc., 1980).

In a supporting developmental toxicity study, conducted according to a protocol similar to OECD Test Guideline 414 but pre-dating GLP, ATMP-H was administered by oral gavage to pregnant CD-1 mice from Day 6 to 15 of gestation at doses of 100, 300 or 1000 mg/kg bw/day. Control animals received equal volumes of distilled water. Females were sacrificed on gestation Day 18 and fetuses were examined for external malformations. Maternal mortality, body weight gains, implantation data, in-life physical observations and gross postmortem examination data, fetal body weights, sex distribution and external malformation data did not reveal any signs of an adverse toxicological effect. No obvious or consistent test substance-related effects on reproduction were observed at any dose. Consequently, the maternal and teratogenicity NOAELs were concluded to be at least 1000 mg/kg bw/day (BioDynamics, 1979d).

In a pilot teratology study, conducted prior to the adoption of OECD test guidelines and pre-GLP, ATMP-H did not cause any adverse effects on dams or fetuses in Long-Evans rats. The rats were administered by oral gavage at doses of 100, 500 or 1000 mg/kg bw/day. No obvious or consistent test substance-related effects on reproduction were observed at any dose. Maternal physical observations and necropsy data as well as fetal gross findings did not reveal evidence of adverse effects. Therefore, maternal and teratogenicity NOAEL were concluded to be at least 1000 mg/kg bw/day in rats (BioDynamics, 1978).

Justification for classification or non-classification

Based on the available read-across data from ATMP-H, no classification is required for ATMP-xNH4 for reproductive or developmental toxicity according to Regulation (EC) No 1272/2008.