Nitrilotrimethylenetris(phosphonic acid)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The key reproductive study is a multigenerational study for the category member, ATMP-H (CAS 6419-19-8, EC No., 229-146-5; 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, Reliability 2).

The reproductive toxicity study (Biodynamics Inc, 1979e, Reliability 2) conducted using the analogue substance DTPMP-H, is included in support of read-across of the OECD 443 study on DTPMP (5 -7Na) to ATMP-H. See the RAAF report for the read-across justification.

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 oestrus cycle, sperm parameters, sexual milestones and no analytical confirmation of exposure levels.

GLP compliance:

no

Remarks:

pre-GLP

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 approximately 370 grams, females approximately 240 grams at mating
- Fasting period before study: No data
- Housing: Individually (except during mating and lactation) in elevated stainless steel wire mesh cages.
- Diet: Ad libitum
- Water: 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: Weekly
- Mixing appropriate amounts with: 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: 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: 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: 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 detailed below

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 were 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 considered to be treatment related deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect on mean body weight or body weight gain during growth or rest periods in males or females. 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):

There was no changes in the food consumption between the dose groups. Further details are available in 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, this was considered as incidental and therefore 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

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. This was considered as not treatment-related changes.

Dermal irritation (if dermal study):

not examined

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):

No changes in the food consumption was observed in any dose groups. Further details are available in 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 in the gross pathological 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):

There was no clinical signs reported.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was a 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 as it did not occur again in other phases of the study and there was no 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 dose-response relationship was present. Generally, these differences 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):

The body weight and weight changes were comparable between all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no difference observed in the food consumption in any of the dose groups.

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):

There was no adverse gross pathological findings in any of the dose groups.

Histopathological findings:

no effects observed

Description (incidence and severity):

There was no adverse histopathological findings in any of the dose groups.

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):

No clinical signs were reported in any of the dose groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was a 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 as it did not occur again in other phases of the study and there was no 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 dose-response relationship was present. Generally, these differences 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):

Body weight and weight changes were comparable between all dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable between all dose groups.

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 were observed (not present in controls and low dose group). This was considered unrelated to treatment by authors as it was not present in other generations. All other necropsy observations were 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 were consistent with minimal to mild interstitial pneumonia and microscopic appearance of the gonads were unremarkable and consistent with sexually immature rats.

Histopathological findings:

no effects observed

Description (incidence and severity):

No histopathological findings were observed.

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 (reliability score 2), conducted before the adaptation to OECD Test Guidelines and pre-GLP, the general and reproductive toxicity NOAEL for ATMP-H (100% active acid) was ≥ 3000 ppm in rats. This is 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

Quality of whole database:

Klimisch score 2

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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 re-mated 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, Reliability 2).

Effects on developmental toxicity

Description of key information

No developmental toxicity data are available on the reproductive toxicity of ATMP-acid, therefore good quality data from the category member, ATMP-xNa (CAS 20592-85-2, EC No., 243-900-0) are read-across.

In the key developmental toxicity study, conducted prior to the adoption of OECD test guidelines and pre-GLP, ATMP-xNa (aqueous solution containing 22.4% w/w neutralised 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 ATMP-xNa/kg bw/day (measured as the neutralised acid). The maternal NOAEL was concluded to be 500 mg/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/kg bw/day respectively. There were no other treatment-related effects observed in the study (BioDynamics Inc., 1979b, Reliability 2).

In the key developmental toxicity study, conducted prior to the adoption of OECD test guidelines and pre-GLP, ATMP-xNa (aqueous solution containing 20% w/w neutralised 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 neutralised acid but this is not clearly specified in the study report. If the doses were in terms of solution as provided, the equivalent atmp-xNa values would be 20, 100, 200 mg/kg bw/day (based on 20% neutralised acid). The NOAEL for maternal toxicity, foetal toxicity and teratogenicity was concluded to be at least 1000 mg/kg bw/day (Biodynamics Inc., 1980 Reliability 2).

Although multiple foetuses from a single high dose (1000 mg/kg bw/day) litter in the rat prenatal toxicity study of ATMP showed a syndrome of findings of the torso, the dam from which this litter was derived showed obvious indications of maternal toxicity. It is therefore concluded that these foetal findings were most likely secondary to maternal effects. All other findings from the foetal examination occurred as single instances and/or without dose-response. In the mouse prenatal toxicity study of ATMP, a single litter at the mid-dose was noted with multiple skeletal findings. Otherwise, limited foetal findings were reported, most without dose-response and/or as single occurrences (Exponent, 2021).

The developmental toxicity study (Monsanto, 1982, Reliability 2) conducted using the analogue substance DTPMP-7Na, is included in support of read-across of the OECD 443 study on DTPMP (5 -7Na) to ATMP-H. See the RAAF report for the read-across justification.

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. 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

Justification for type of information:

See attachment to Section 13 for justification of read-across for the ATMP category (relevant for read across from ATMP-xNa to ATMP-H).

Reason / purpose for cross-reference:

read-across: supporting information

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

Dose / conc.:

500 mg/kg bw/day

Remarks:

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

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

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

Based on:

test mat.

Remarks:

Assumed to be as neutralised acid (ATMP-xNa) but not clearly specified in the study report. If the dose was in terms of solution as provided, the equivalent value would be 200 mg/kg bw/day (based on 20% neutralised 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 neutralised acid (ATMP-xNa) but not clearly specified in the study report. If the dose was in terms of solution as provided, the equivalent value would be 200 mg/kg bw/day (based on 20% neutralised 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 2) conducted prior to the adoption of OECD test guidelines and GLP, it was concluded that ATMP-xNa (aqueous solution containing 20% w/w neutralised 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, fetal toxicity and teratogenicity was greater than 1000 mg/kg bw/day. The doses are assumed to be as neutralised acid but this is not clearly specified in the study report. If the doses were in terms of solution as provided, the equivalent ATMP-xNa values would be 20, 100, 200 mg/kg bw/day (based on 20% neutralised acid).

Reference 2

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

Justification for type of information:

See attachment to Section 13 for justification of read-across for the ATMP category (relevant for read across from ATMP-xNa to ATMP-H).

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

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:

no

Remarks:

pre-GLP

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Wilmington, Mass
- Age: 72 days at mating
- Weight at study initiation: Approximately 240 - 250 grams
- Fasting period before study: Not specified
- Housing: Individually housed
- Diet: Purina Certified Rodent Chow 5002, ad libitum
- Water: Ad libitum
- Acclimation period: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not specified
- Humidity (%): Not specified
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): Not specified

IN-LIFE DATES: Not specified

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance was mixed with water and administered at 10 mg/kg bw/day. Volume was adjusted based on the most recent bodyweight data and was prepared daily.

VEHICLE
- Justification for use and choice of vehicle: Not specified
- Concentration in vehicle: Not specified
- Amount of vehicle: Not specified
- Lot/batch no.: N/a
- Purity: N/a

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.:

0 mg/kg bw/day

Remarks:

Control group

Dose / conc.:

100 mg/kg bw/day

Remarks:

measured as the neutralised acid (ATMP-xNa)

Dose / conc.:

500 mg/kg bw/day

Remarks:

measured as the neutralised acid (ATMP-xNa)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

measured as the neutralised acid (ATMP-xNa)

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 days
Other: Dams were sacrified on GD 21 by lethal exposure to diethyl ether

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: GD 0, 6, 10, 15, 20 and 21 (pre-necropsy)

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 6 -15, 21

FOOD CONSUMPTION AND COMPOUND INTAKE: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Not specified

OTHER: Dams

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: N/a

Blood sampling:

- Plasma: No
- Serum: No
- Volume collected : N/a
- Other: N/a

Fetal examinations:

- External examinations: Yes: Half per litter
- Soft tissue examinations: Yes: Half per litter
- Skeletal examinations: Yes: Half per litter (staining used: Alizarin red).
- Head examinations: Yes: Half per litter - Wilson serial sectioning (10X or 20X magnification) after fixing in Bouin's solution
- Anogenital distance of all live rodent pups: Yes (crown-rump distance)

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 were observed in any dose group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

One female from the 100 mg/kg bw/day dose group was in moribund condition and sacrificed on GD 6 (first day of treatment).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant differences in maternal body weight gain were observed between the groups. The body weight gain in the 1000 mg/kg bw/day dose group was slightly lower than for the other dose groups, however, not statistically significant lower. This was considered to depend on an individual dam that had a lower body weight gain than the others.

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:

effects observed, non-treatment-related

Description (incidence and severity):

There were a few adverse changes present, however, no treatment-related effects.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The implantation loss were statistically significantly lower in the 100 mg/kg bw/day dose group, however, this was considered as not a treatment-related change as no dose-response relationship could be observed.

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Description (incidence and severity):

All values were within the historical control range for all dose groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were obtained in any dose groups.

Changes in pregnancy duration:

not examined

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/day). This decrease was statistically significant, however, considered as not treatment-related changes no dose-response relationship could be observed.

Other effects:

no effects observed

Description (incidence and severity):

The mean number of corpora lutea was comparable in all dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

act. ingr.

Remarks:

neutralised acid (ATMP-xNa)

Basis for effect level:

body weight and weight gain

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No significant differences in the body weight were observed between the dose groups.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no reduction in the number of live offspring in any dose groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no statistically significant effect in the sex ratio between the dose groups.

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The crown-rump length in males were: 4.2/4.2/4.3 (P<0.01)/4.2 and in females: 4.1/4.1/4.2 (P<0.01)/4.1. In the 500 mg/kg bw/day dose group, an increase was observed, however, this was not considered as biologically significant as the change did not occur in a dose-response relationship.

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. This was considered as incidental as all the remaining foetuses in this dose group was unremarkable as was all the other dose groups.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There was no statistically significant effect in the variation in ossification between the dose groups.
The total foetuses examined for skeletal malformations were: 177/158/169/159 and per foetus: 4.0%/1.9%/3.0%/1.3% and per litter: 20.8%/13.6%/20.8%/8.7%. There was no significant effect. In the control group, skeletal malformations included angulated ribs, cervical rib and wavy rib. In the 100 mg/kg bw/day dose group, angulated rib, cervical rib, 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. In the 1000 mg/kg bw/day, 5 lumbar vertebra and fused sternebrae were observed. The occurrences of skeletal malformations were considered as not treatment-related changes.

Visceral malformations:

no effects observed

Description (incidence and severity):

In the control group, distended renal pelvis, renal pelvis, distended ureter and bladder were observed. In addition, the 100 mg/kg bw/day dose group, a fold in the retina and distended ureter with or without a distended renal pelvis were observed. In the 500 mg/kg bw/day, distended ureter and an ectopic kidney were observed. In the 1000 mg/kg bw/day, a fold in the retina, anophthalmia, a malposition of the testis and malrotation of the heart were observed. The malrotation of the heart occurred in two foetuses in the 1000 mg/kg bw/day from two different litters. However, the changes were considered as incidental as they occurred similarly across all the dose groups.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

neutralised acid (ATMP-xNa)

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:

neutralised acid (ATMP-xNa)

Sex:

male/female

Basis for effect level:

other: No foetotoxic effects observed.

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

In a well-documented teratology study (reliability 2), conducted prior to the adaptation to OECD Test Guidelines and pre-GLP, ATMP-xNa (aqueous solution containing 22.4% w/w neutralised acid) was not embryotoxic or teratogenic when administered to rats at 100 or 500 mg/kg bw/day (measured as the neutralised acid) by gavage on GD6-15. At 1000 mg/kg bw/day, six foetuses 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 foetuses were normal. The clear absence of any comparable effect in other high dose litters and lack of dose-response, indicates that 1000 mg ATMP-xNa/kg bw/day was the no-effect level for embryotoxicity and foetotoxicity. The maternal NOAEL was concluded to be 500 mg ATMP-xNa/kg bw/day.

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

Quality of whole database:

Klimisch score 2

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

In the key developmental toxicity study (BioDynamics Inc, 1979b), ATMP-xNa (aqueous solution containing 22.4% w/w neutralised 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/kg bw/day (measured as the neutralised 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. The maternal NOAEL was concluded to be 500 mg/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/kg bw/day respectively. There were no other treatment-related effects observed in the study (BioDynamics Inc., 1979b, Reliability 2).

The Registrants commissioned an independent expert review of this study (Exponent, 2021; see attachment for full report). The findings from this expert review are as follows:

Dams at 1000 mg/kg bw/day gained 12% less body weight over the treatment period (GD 6 to GD 15) than did controls. No other potential indications of maternal toxicity were evident in the study. No effect of treatment on pregnancy and litter parameters was observed (Table 1). While the number of corpora lutea at 100 mg/kg bw/day was significantly lower than control, because the egg is released prior to the onset of dosing, this finding is unrelated to treatment. Although the mean numbers of resorptions were higher in the treated groups compared to control (statistically significant at 100 and 1000 mg/kg bw/day), these findings did not show a dose-response and the finding at 1000 mg/kw bw/day was driven in part by a whole litter resorption experienced by one dam at this dose. Crown-to-rump length (a measure not typically taken today) was statistically increased for both male and female foetuses at 500 mg/kg bw/day (data not shown). This finding is consistent with the non-statistically increased foetal body weights (a more sensitive indicator than crown-to-rump lengths) observed at this dose compared to control. The difference did not show a dose-response was considered non-adverse and is unlikely to be related to ATMP treatment.

Table 1. Pregnancy & litter parameters from the rat prenatal toxicity study of ATMP.

Doses (mg/kg bw/day:	0	100	500	1000
No. pregnant females	23	22	24	24
No. corpora lutea	17.2 ± 2.8	16.0* ± 1.7	16.7 ± 3.6	16.8 ± 3.9
No. implantations	14.5 ± 2.6	14.7 ± 2.6	14.0 ± 2.2	13.7 ± 3.2
No. resorptions	0.2 ± 0.4	0.8* ± 1.3	0.5 ± 0.8	0.8* ± 1.3
No. foetuses	14.3 ± 2.6	13.9 ± 3.2	13.5 ± 2.4	12.9 ± 3.8
Sex ratio^a	0.92	1.02	0.94	0.87
Foetal wt. – M	5.54 ± 0.30	5.43 ± 0.77	5.71 ± 0.33	5.49 ± 0.40
Foetal wt. – F	5.25 ± 0.35	5.17 ± 0.73	5.34 ± 0.30	5.16 ± 0.39

* p<0.05

^aRatio of total male to total female foetuses per group.

All foetal external and visceral findings were reported as “observations”; skeletal findings were called “malformations”. No variations were noted. At the foetal external examination, two and one foetuses at 500 and 1000 mg/kg bw/day, respectively, showed blood blisters in the scapular region. These are likely a result of the handling of the foetal specimens and unrelated to treatment; thus, they were not included in the totals reported in the study report summary data and are not detailed in Table 2 below. One or two foetuses per dose group (including control) were reported at the external examination (sic) as having distended ureter and/or distended renal pelvis. It is unclear why these findings were categorized as external findings since they are all internal soft tissue findings and more appropriately should have been noted as part of the visceral examination instead. Similar findings involving the ureter, renal pelvis, bladder and kidneys were noted in all dose groups at the visceral examination. These findings showed no relation to treatment and have been determined to be transient findings associated with developmental delay (Woo and Hoar, 1972). Thus, they are not considered to be adverse.

Six foetuses from Litter 822 were reported to have multiple findings at the external examination; these are detailed in Table 3. Some observations appear to be different descriptions of the same endpoint (e.g., the trunk being shortened and the distance between the umbilicus and sternum being reduced; forward flexure of the head and tension of the skin not allowing normal head extension). Further, several descriptions are unclear (e.g., distension of the abdominal-thoracic region; constriction of the hindleg at the hock). Additional observations reported in foetuses from this litter (as indicated by the ‘b’ footnote in Table 2) included: testes at posterior end of kidneys (foetus #822-5), heart skewed 30⁰ (foetuses #822-11 & #822-13), and sternebrae fused (foetus #822-5). The exact meaning of the description “heart skewed 30⁰” is not clear; however, it is important to note that, because this finding was observed in two of the foetuses with multiple findings of the trunk, its association with the phenomenon responsible for these trunk findings cannot be discounted.

Importantly, dam #822 gained only 22 grams over the treatment period (GD 6 – GD 15); this was the lowest body weight gain during the dosing period of all dams in the high dose group and the 3rd lowest of all dams on study. The foetal findings (either singly or together) were not observed in any other foetus in the study, suggesting that these observations were not due to a direct foetal effect of treatment. Further examination of the study data demonstrated that the reduced maternal weight gain was not the result of reduced foetal weights or reduced litter size, indicating that the dam experienced maternal toxicity, which would have led to secondary effects on her litter. To understand why multiple foetuses in the litter shared the same suite of observations, it is important to recognize that there is a tendency for foetuses within a given litter to exhibit similar morphologies and endpoints (US EPA, 1991). This tendency is known as the "litter effect". When there is a litter effect, it can fallaciously increase the apparent incidence of a particular finding. Litter effects are thought to be caused in large part by the shared genetics of littermates and the fact that all foetuses of a given litter are exposed to the same maternal environment. These two factors account for the similarity in outcomes within that litter, whereas the different genetics and maternal environments that occur from litter to litter within the remainder of the litters from the same treatment group can explain the absence of findings in those litters (Holson and Pearce, 1992; Festing, 2006). Consequently, it is likely that the constellation of foetal findings in litter #822 is secondary to maternal toxicity and is not a direct developmental toxicity effect.

The two dams that gained less weight over the treatment period showed microscopic findings consistent with disease: #220 (control group; gained 21 grams) exhibited cyst-like foci in the lungs and #403 (low dose group; gained 8 grams) showed inflammation of the salivary glands, thymus, spleen and lymph nodes in conjunction with material consistent with infection).

All other findings from the foetal examination occurred as single instances and/or without dose-response (and in some cases, were not observed in the high dose group); thus, they are most likely unrelated to treatment.

Table 2. Foetal malformations data from the rat prenatal toxicity study of ATMP-xNa (BioDynamics Inc, 1979b).

Doses (mg/kg bw/day:	0	100	500	1000
EXTERNAL EXAMINATION
Total foetuses (litters) examined:	177 (24)	158 (22)	169 (24)	159 (24)
Total foetuses (litters) with external malformations:	1 (1) 0.6 (4.2)^a	2 (2) 1.3 (9.1)	1 (1) 0.6 (4.2)	8 (3) 5.0 (12.5)
Multiple findings	---	---	---	6 (1)^b 3.8 (4.2)
Ureter distended^b	1 (1) 0.6 (4.2)	2 (2) 1.3 (9.1)	1 (1) 0.6 (4.2)	2 (2) 1.3 (8.3)
Renal pelvis distended^b	---	1 (1) 0.6 (4.5)	---	---
Testes at posterior ends of kidneys^b	---	---	---	1 (1)^b 0.6 (4.2)
VISCERAL EXAMINATION
Total foetuses (litters) examined:	152 (24)	147 (22)	156 (24)	150 (24)
Total foetuses (litters) with external malformations:	7 (4) 4.3 (16.7)^a	12 (9) 8.2 (40.9)	7 (7) 4.5 (29.2)	6 (5) 4.0 (20.8)
Distended renal pelvis	3 (3) 2.0 (12.5)	9 (6) 6.1 (2.7)	5 (5) 3.2 (20.8)	1 (1) 0.7 (4.2)
Distended ureter	2 (2) 1.3 (8.3)	3 (3) 0.2 (13.6)	2 (2) 1.3 (8.3)	2 (2) 1.3 (8.3)
Distended bladder	4 (1) 2.6 (4.2)	1 (1) 0.7 (4.5)	---	---
Ectopic kidney	---	---	1 (1) 0.6 (4.2)	---
Retinal fold	---	1 (1) 0.7 (4.5)	---	1 (1) 0.7 (4.2)
Anophthalmia	---	---	---	1 (1) 0.7 (4.2)
Heart skewed 30⁰	---	---	---	2 (1)^b 1.3 (4.2)
SKELETAL EXAMINATION
Total foetuses (litters) examined:	177 (24)	158 (22)	169 (24)	159 (23)
Total foetuses (litters) with external malformations:	7 (5) 4.0 (20.8)^a	3 (3) 1.9 (13.6)	5 (5) 3.0 (20.8)	2 (2) 1.3 (8.7)
Angulated ribs	2 (1) 1.1 (4.2)	2 (2) 1.3 (9.1)	1 (1) 0.6 (4.2)	---
Wavy ribs	1 (1) 0.6 (4.2)	1 (1) 0.6 (4.5)	1 (1) 0.6 (4.2)	---
7^thCervical rib	4 (4) 2.3 (16.7)	1 (1) 0.6 (4.5)	3 (3) 1.8 (12.5)	---
7^thLumbar vertebrae	---	---	1 (1) 0.6 (4.2)	---
5 Lumbar vertebrae	---	---	---	1 (1) 0.6 (4.3)
Sternebrae fused	---	---	---	1 (1)^b 0.6 (4.3)

^aPercent affected foetuses (percent affected litters)

^bThis finding of the soft tissue is more appropriately considered a visceral finding, but for the purposes of presenting the data as provided in the study report, the finding is listed as an external examination finding.

Table 3. The multiple findings noted in foetuses from litter #822 at the foetal external examination.

Litter 822	1	5	9	11	13	16
Both forepaws slightly flexed	X	X	X	X	X	X
Distance between umbilicus and sternum slightly reduced	X	X	X	X	X	X
Trunk shortened and thickened	X	X	X	X	X	X
Exaggerated forward flexure of head	X	X	X	X	X	X
Tension of skin from mandible to chest would not permit normal extension of head	X	X	X	X	X	X
Distance between umbilicus and genitalia increased			X
Lateral abdominal-thoracic areas moderately distended		X		X	X	X
Slight constriction of right/left hind leg at hock				X	X

In the key developmental toxicity study, ATMP-xNa (aqueous solution containing 20% w/w neutralised 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 neutralised acid but this is not clearly specified in the study report. Parameters for evaluation included mortality, body weight, clinical signs and uterine implantation data, ossification variation data and teratological evaluation (BioDynamics Inc., 1980, Reliability 2).

The Registrants commissioned an independent expert review of this study (Exponent, 2021; see attachment for full report). The findings from this expert review are as follows:

No potential indications of maternal toxicity were evident in the study. Pregnancy and litter parameters are shown in Table 4; note that numbers of corpora lutea were not reported. No effect of ATMP was observed on pregnancy outcomes. As in the rat study, all foetal external and visceral findings were reported as “observations”; skeletal findings were called “malformations”. Findings from the foetal examinations are shown in Table 5. Three foetuses from two litters at 500 mg/kg bw/day were identified with exencephaly; two of these also displayed open eye, a finding that common coincides with exencephaly. Importantly, exencephaly was not seen at the highest dose tested – 1000 mg/kg bw/day – and thus is unlikely to be related to treatment.

Cleft palate was noted in one foetus at 1000 mg/kg bw/day at the external examination (in combination with open eye) and a single foetus at 100 mg/kg bw/day at the visceral examination. This latter finding, because it was not seen at the external examination, was likely a partial, posterior cleft of the soft palate. Cleft palate is a relatively common malformation in mice. The study report notes a historical incidence of cleft palate in CD-1 mice of 0.5%. More recently, a mean foetal incidence of 0.34% (range: 0 -2.54%) has been reported for CD-1 mice evaluated at Charles River Laboratories’ Ashland site (Coder, 2017). Because of this, and the fact that it did not occur with a dose-response, it is unlikely that this finding is related to ATMP treatment.

Several observations involved positional orientation of the limbs with no underlying skeletal changes. “Hindlimb flexures” were observed in 2-3 foetuses in each of the ATMP-treated groups, but not in the control group; one of these foetuses was also noted to have leg and foot turned inward. An additional two foetuses – one each in the 500 and 1000 mg/kg bw/day dose groups – were noted to have “hindlimb extensions defects”. These descriptors are ambiguous and how the two findings differ from each other is not clear. Nevertheless, muscle contracture of the limbs in the absence of skeletal defects affecting the joints are typically transient findings that self-correct (DeSesso and Scialli, 2018). Importantly, these findings show no relation to findings noted at the skeletal examination. Further, the combined incidence of these muscle contracture findings did not show a relation to treatment.

Table 4. Pregnancy & litter parameters from the mouse prenatal toxicity study of ATMP.

Doses (mg/kg bw/day:	0	100	500	1000
No. pregnant females	24	28	34	32
No. implantations	12.0 ± 1.7	11.4 ± 2.6	11.8 ± 2.3	12.1 ± 1.7
No. resorptions	1.0 ± 1.3	0.7 ± 0.9	0.4 ± 0.7	1.0 ± 1.0
No. live foetuses	11.0 ± 1.8	10.6 ± 2.7	11.2 ± 2.1	11.0 ± 2.0
No. dead foetuses	0.0 ± 0.0	0.04 ± 0.2	0.1 ± 0.3	0.1 ± 0.3
Sex ratio^a	1.05	1.26	1.15	1.19
Foetal wt. – M	1.33 ± 0.08	1.34 ± 0.11	1.34 ± 0.11	1.35 ± 0.08
Foetal wt. – F	1.29 ± 0.09	1.30 ± 0.09	1.27 ± 0.10	1.30 ± 0.09

^aRatio of total male to total female foetuses per group.

Table 5. Foetal malformations data from the mouse prenatal toxicity study of ATMP.

Doses (mg/kg bw/day:	0	100	500	1000
EXTERNAL EXAMINATION
Total foetuses (litters) examined:	263 (24)	299 (28)	386 (34)	355 (32)
Total foetuses (litters) with external malformations:	1 (1) 0.4 (4.2)^a	3 (3) 1.0 (10.7)	7 (6) 1.8 (17.6)	6 (6) 1.7 (18.8)
Exencephaly	---	---	3 (2) 0.8 (8.8)	---
Eye open	1 (1) 0.4 (4.2)	---	2 (2) 0.5 (5.9)	1 (1) 0.3 (3.1)
Cleft palate	---	---	---	1 (1) 0.3 (3.1)
Hindlimb flexure	---	3 (3) 1.0 (10.7)	2 (2) 0.5 (5.9)	2 (2) 0.6 (6.3)
Hindlimb extension defect	---	---	1 (1) 0.3 (2.9)	1 (1) 0.3 (3.1)
Discoloration on tail	---	---	1 (1) 0.3 (2.9)	2 (2) 0.6 (6.3)
VISCERAL EXAMINATION
Total foetuses (litters) examined:	125 (24)	143 (28)	183 (34)	171 (32)
Total foetuses (litters) with visceral malformations:	1 (1) 0.8 (4.2)^a	1 (1) 0.7 (3.6)	2 (2) 1.1 (5.9)	1 (1) 0.6 (3.1)
Exencephaly	---	---	1 (1)^b 0.5 (2.9)	---
Eye open	1 (1)^b 0.8 (4.2)	---	---	---
Eye – lens out of optic cup, fold in retina	---	---	---	1 (1) 0.6 (3.1)
Cleft palate	---	1 (1) 0.7 (3.6)	---	---
Distended renal pelvis	---	---	1 (1) 0.5 (2.9)	---
SKELETAL EXAMINATION
Total foetuses (litters) examined:	138 (24)	156 (28)	203 (34)	183 (32)
Total foetuses (litters) with skeletal malformations:	0 (0) 0.0 (0.0)^a	1 (1) 0.6 (3.6)	9 (3) 4.4* (8.8)	2 (2) 1.1 (6.3)
Misshapen tibia and/or fibia	---	---	5 (1) 2.5 (2.9)	---
Angulated ribs	---	---	5 (1) 2.5 (2.9)	---
7^thCervical transverse process missing	---	1 (1) 0.6 (3.6)	---	---
2^nd& 3^rdtransverse process fused	---	---	---	1 (1) 0.5 (3.1)
Cervical rib	---	---	1 (1) 0.5 (2.9)	---
5 Lumbar vertebrae	---	---	2 (1) 1.0 (2.9)	---
Sternebrae fused	---	---	1 (1) 0.5 (2.9)	---
Sternebrae scrambled	---	---	2 (2) 1.0 (5.9)	1 (1) 0.5 (3.1)

^aPercent affected foetuses (percent affected litters)

^bConfirmation of findings noted at external examination.

All other findings from the foetal examination occurred at single instances and/or without dose-response. In some cases, the findings were not observed in the high dose group. Consequently, their relation to treatment is unlikely. It should be noted, however, that foetuses in Litter #604 from the middle dose group exhibited multiple skeletal anomalies (Table 6). Litter #604 had the smallest foetuses on the study, weighing 20-25% less than the group average. The foetuses from this litter accounted for almost all of the skeletal anomalies noted at 500 mg/kg bw/day. Because of the findings occurred in a single litter and did not repeat at the highest dose tested, they were appropriately considered not related to treatment.

 

Table 6. The multiple findings noted in foetuses from litter #604 at the foetal skeletal examination.

Litter 604	1	3	5	7	9	11
Tibia misshapen	X	X	X	X		X
Fibula misshapen		X		X		X
Ribs angulated		X	X	X	X	X
Sternebrae fused (4^th& 5^th)				X
Sternebrae scrambled				X	X

In a supporting developmental toxicity study, conducted according to a protocol similar to OECD Test Guideline 414 but pre-dating GLP, ATMP-xNa 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, Reliability 2).

In a pilot teratology study, conducted prior to the adoption of OECD test guidelines and pre-GLP, ATMP-xNa 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, Reliability 2).

Justification for classification or non-classification

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