2-amino-2-methylpropanol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Remarks:

definitive study OECD 414 in rabbits

Principles of method if other than guideline:

preliminary study before OECD 414 in rabbits

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

AMP-95, industrial grade

Species:

rabbit

Strain:

New Zealand White

Sex:

female

Route of administration:

oral: gavage

Details on oral exposure:

Group 2 (2 mg/mL), Group 3 (5 mg/mL), and Group 4 (15 mg/mL) formulations were prepared three times by adding the required amount of test substance to the appropriate amount of the vehicle/control substance and mixing until visually clear. Formulations were adjusted for pH using NaOH or HCl. The final pH ranged from 6.90-7.50.
The animals were dosed by via oral gavage once daily at a volume of 5 mL/kg for 22 consecutive days. Dosing volumes were based on the animals’ most recent body weights.

Duration of treatment / exposure:

22 days

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

containing 9.5 mg AMP/kg bw/day, taking into account 5.4% water content

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

containing 23.7 mg AMP/kg bw/day, taking into account 5.4% water content

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

containing 71.0 mg AMP/kg bw/day, taking into account 5.4% water content

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two group 4 females at 75 mg/kg/day showed low food consumption/anorexia, body weight loss, and few feces.

Mortality:

mortality observed, treatment-related

Description (incidence):

One of the females with clinical signs was euthanized as moribund on SD 19.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights and body weight gains were decreased at 75 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was decreased at 75 mg/kg/day.

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:

no effects observed

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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

other: maximum tolerated dose, MTD

Effect level:

>= 25 - < 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

containing 23.7-71.0 mg AMP/kg bw/day, taking into account 5.4% water content

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

Conclusions:

In a 3-week dose range finding study in female rabbits, with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, the maximum tolerated dose (MTD) ranged >=23.7 to <71.0 mg AMP/kg bw/day.

Executive summary:

In a 3-week dose range finding study in female rabbits treated by oral gavage:

• At 71.0 mg AMP/kg bw/day, two females showed low food consumption/anorexia, body weight loss, and few feces. One of them was euthanized as moribund on SD 19. Mean body weights, body weight gains and food consumption were decreased.


• At 9.5 and 23.7 mg AMP/kg bw/day, no adverse effects were noted. 

Below conclusions were drawn post-report by the registrant:

1) Based on the results, AMP's maximum tolerated dose (MTD) ranges >=23.7 to <71.0 mg AMP/kg bw/day.

2) A NOAEL was reported, but should be ignored as no liver histopathology was performed (target organ).

3) At the toxic dose of 71.0 mg AMP/kg bw/day, AMP concentration in dosage form was 15 mg/mL i.e. 1.5% w/w. AMP was tested neutralized to pH 6.9-7.5 using HCl. As is, AMP is alkaline (industrial-grade: pKa = 9.74). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3309 ln(Concentration in % w/w) + 11.548], pH of industrial-grade AMP solutions at 1.5 % AMP w/w would be 11.7. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is, the study's toxic dose could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines referenced by REACH do NOT require any neutralization or pH adjustment of test items, this study represents artificial dose maximization in excess of REACH principles, removing AMP's critical toxic effect which is pH-dependent non-specific toxicity.

4) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

2-amino-2-methylpropanol

EC Number:

204-709-8

EC Name:

2-amino-2-methylpropanol

Cas Number:

124-68-5

Molecular formula:

C4H11NO

IUPAC Name:

2-amino-2-methylpropan-1-ol

Specific details on test material used for the study:

AMP-95: industrial grade

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Text Table 3: Animal Information
Females
Species and Strain Time-mated female New Zealand White Rabbits
Supplier Charles River Breeding Labs; Saint Constant, Canada
Method of Identification Cage Card/Tattoo
Number of Animals Received 82
Number Used on Study 80
Age at First Dose 4.6 – 6.2 months
Weight Range at Mating 3245 – 4499 g
Weight Range at First Dose 3164.9 – 4352.1 g

Animals were acclimated to laboratory conditions for at least three days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance, AMP-95, was supplied as an 882 mg AMP/mL solution with an expiration date of 06 April 2024. The test substance was received at ambient temperature and stored at room temperature upon receipt. The control substance, DI water, was received from a vendor/supplier and stored at room temperature upon receipt. Certificates of Analysis are presented in Appendix 1. Reserve samples (1 mL) of the neat test substance and control substance were taken and subsequently archived under the same storage conditions as those of the neat materials.
The neat test and vehicle/control substance were considered 100% pure for formulation purposes.
The vehicle/control article, DI water, was used as received and adjusted for pH using NaOH or HCl on each day of dosing. Final pH ranged from 6.96-7.42. Formulations were stored at room temperature until used for dosing.
Group 2 (6 mg/mL), Group 3 (20 mg/mL), and Group 4 (60 mg/mL) formulations were prepared daily, with the exception of the last preparation, by adding the required amount of test substance to the appropriate amount of the vehicle/control substance and mixing until visually clear. Formulations were adjusted for pH using 1N NaOH and/or HCl. The final pH ranged from 6.64-7.47. Additional vehicle was added until the required final volume was reached. Formulations were kept at room temperature until used for dosing on the day of preparation. The last preparation was assigned a 7-day shelf life and stored at room temperature until used for dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were sampled and stored.
Stability analysis of the dose formulations was performed under Inotiv Study No. PRO.1000-192139-001 as part of the method validation.
One set of samples was shipped (on dry ice) to the Inotiv West Lafayette for test substance concentration analysis. Following receipt of analytical results, unused samples were discarded with authorization from the Study Director.

Duration of treatment / exposure:

The animals were dosed once daily from GD 7 to 28 (day of confirmation of mating = GD 0) via oral gavage at a volume of 5 mL/kg. Dose volumes were based on the most recent body weights.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

contains 9.5 mg/kg bw/day AMP, taking into account 5.4% water content

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

contains 23.7 mg/kg bw/day AMP, taking into account 5.4% water content

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

contains 71.0 mg/kg bw/day AMP, taking into account 5.4% water content

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2 or more then 2 times daily
- Cage side observations checked in table [Yes] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On presumed GD 29

BODY WEIGHT: Yes
- Time schedule for examinations:GD 7, 10, 13, 16, 19, 21, 24, and 27
Prior to necropsy (GD 29)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined:
Esophagus,Trachea,Lungs,Uterus,parathyroid & thyroid gland, spleen,stomach, thymus,Kidney, Intestine,Ovary,Pancreas,mammary gland, heart and liver
OTHER: liver weight, gravid uterine weight

Ovaries and uterine content:

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

Fetal examinations:

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

Statistics:

ANOVA
Shapiro-Wilk test
Levene’s test
Dunnett’s t-test

Indices:

Percent Pre-implantation Loss = [(total number of corpora lutea – total number of implantations) / total
number of corpora lutea] X 100
Percent Post-implantation Loss = (total post-implantation loss / total number of implantations) X 100

Historical control data:

Persistent truncus arteriosus has been noted in a larger developmental and reproductive historical control data base from Charles River Ashland (mean 0.03 % per litter, maximum 0.7% per litter), and has been observed as a spontaneous malformation in rabbits (Hood, 2012). It is considered to be a heritable trait and the genetic makeup of both parents could influence the penetrance and/or spontaneous observation (Hood, 2012). Due to the low incidence and absence of any other evidence of maternal or developmental toxicity at
≤ 25 mg/kg/day, this malformation was not considered test substance related.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at 75 mg/kg/day: adverse clinical observations of red/bloody discharge (from vagina, nose), few feces, pale, audible respiration, and languid (hypoactivity)

Dermal irritation (if dermal study):

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

at 75 mg/kg/day: only one rabbit survived until schedule termination; all other rabbits were euthanized as moribund (17/20) or found dead (2/20).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at 75 mg/kg/day: body weight loss (mean body weights 0.93x controls on GD 21)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at 75 mg/kg/day: decreased food consumption (approximately 30 grams vs. 110 in controls on GD 20-21)

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:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One female at 25 mg/kg/day and 11 females at 75 mg/kg/day had discolored liver, described as pale tan, or tan.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

75 mg/kg/day: minimal to moderate liver vacuolar degeneration/necrosis + minimal to moderate liver hepatocellular periportal macrovesicular vacuolation + mild to moderate multifocal liver necrosis + minimal liver ductular hyperplasia
25 mg/kg/day: minimal to mild liver hepatocellular periportal macrovesicular vacuolation + minimal ductular hyperplasia
10 mg/kg/day: minimal liver hepatocellular periportal macrovesicular vacuolation in 1/20 animals only

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

The top-dose of 75 mg/kg/day largely exceeded the MTD based notably on mortality in 19/20 rabbits. Therefore effects in this group cannot be used to assess reproductive and developmental effects.

Maternal developmental toxicity

Number of abortions:

not specified

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination and therefore the effect of AMP-95 on fetal survival (abortions) could not be assessed on GD 29.
No effect at lower doses.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination. In this animal there were fewer implantation sites (8 vs. 10.9 in controls), with 27% pre-implantation loss and 0% post-implantation loss.
No effect at lower doses.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination. At unscheduled necropsies, there were six rabbits with total litter loss consisting of primarily late resorptions (compared to 0 in controls). This was due to low food consumption.
No effect at lower doses.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination. At unscheduled necropsies, there were
- 6 rabbits with total litter loss consisting of primarily late resorptions (compared to 0 in controls)
- only 1 rabbit with live fetuses
This was due to low food consumption.
No effect at lower doses.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day, only one animal survived to scheduled termination. At unscheduled necropsies, there were nine dead fetuses in three litters (compared to 0 in controls). This was due to low food consumption.
No effect at lower doses.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The top-dose of 75 mg/kg/day largely exceeded the MTD based notably on mortality in 19/20 rabbits. Therefore effects in this group cannot be used to assess reproductive and developmental effects.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

contains 23.7 mg/kg bw/day AMP, taking into account 5.4% water content

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

pre and post implantation loss

total litter losses by resorption

2

Key result

Dose descriptor:

LOAEL

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

contains 71.0 mg/kg bw/day AMP, taking into account 5.4% water content

Basis for effect level:

body weight and weight gain

clinical signs

dead fetuses

early or late resorptions

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

pre and post implantation loss

total litter losses by resorption

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not specified

Description (incidence and severity):

75 mg/kg/day: Slightly lower mean fetal weight (34.9 g vs. 38.8 g in controls); statistical and biological significance cannot be determined due to small sample size (only 1 litter and 8 fetuses)
No effect at lower doses

Reduction in number of live offspring:

not specified

Description (incidence and severity):

75 mg/kg/day: Slightly lower number of live fetuses per litter (8 vs. mean of 10.8 in controls); statistical and biological significance cannot be determined due to small sample size (only 1 litter and 8 fetuses)
No effect at lower doses

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Description (incidence and severity):

75 mg/kg/day: Slightly lower litter weight (279.3 g vs. mean of 412.8 g in controls); statistical and biological significance cannot be determined due to small sample size (only 1 litter and 8 fetuses)
No effect at lower doses

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

The top-dose of 75 mg/kg/day largely exceeded the MTD based notably on mortality in 19/20 rabbits. Therefore effects in this group cannot be used to assess reproductive and developmental effects. There were some minimal growth/survival effects at the top-dose (lower fetal and litter weights, less live fetus) but statistical and biological significance cannot be determined due to small sample size (only 1 litter and 8 fetuses); effects would be attributable to the excess maternal toxicity.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

contains 23.7 mg/kg bw/day AMP, taking into account 5.4% water content

Sex:

male/female

Remarks on result:

other: the next higher dose resulted in so high maternal toxicity that the relevance of the growth/survival effects observed (lower fetal and litter weight, less live fetuses) cannot be judged

Remarks:

maternal no observed adverse-effect level (NOAEL) was determined to be 25 mg/kg/day and the fetal NOAEL was determined to be 25 mg/kg/day.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a rabbit oral developmental toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, 71.0 mg AMP/kg bw/day led to excess maternal toxicity triggering a cascade of non-specific developmental effects.
The NOAEL was 23.7 mg AMP/kg bw/day for maternal and developmental effects.

Executive summary:

In an OECD 414 study, pregnant New Zealand White rabbits were orally gavaged with AMP-95 (neutralized to pH 6.6-7.5) at dose levels of 0, 10, 25 or 75 mg/kg bw/day over GD 7-28 (containing 9.5, 23.7 and 71.0 mg AMP/kg bw/day resp., taking into account 5.4% water content). Surviving does were subjected to a gross necropsy,and uterine and fetal examinations on presumed GD 29.

• 71.0 mg AMP/kg bw/day largely exceeded the MTD. Mortality occured in 19/20 rabbits. Red/bloody discharge (from vagina, nose), few feces, pale, audible respiration, and languid (hypoactivity) were observed. Body weight loss occured (-17%) due to markedly reduced food consumption (-73% on GD 20). 11 rabbits had discolored liver, and there were multiple adverse liver lesions (minimal to moderate liver vacuolar degeneration/necrosis + minimal to moderate liver hepatocellular periportal macrovesicular vacuolation + mild to moderate multifocal liver necrosis + minimal liver ductular hyperplasia). The surviving rabbit had fewer implantation sites (8 vs. 10.9 in controls), with 27% pre-implantation loss and 0% post-implantation loss. Several decedent rabbits showed total litter loss consisting of primarily late resorptions and dead fetuses were noted. Mean fetal weight, litter weight and number of live fetuses per litter were slightly lower in the litter from the surviving rabbit. Statistical and biological significance of developmental effects cannot be determined, but they can be considered seconadry to the very high maternal toxicity.


• At 23.7 mg AMP/kg bw/day, one rabbit had discolored liver, and liver lesions were non-adverse (minimal to mild liver hepatocellular periportal macrovesicular vacuolation + minimal ductular hyperplasia). No developmental effects.


• At 9.5 mg AMP/kg bw/day, liver lesions were non-adverse (minimal liver hepatocellular periportal macrovesicular vacuolation in 1 animal only). No developmental effects.

Under the conditions of this study, the NOAEL for maternal toxicity was 23.7 mg/kg bw/day. The NOAEL for developmental toxicity was 23.7 mg/kg bw/day, the highest dose level without excess maternal toxicity.

Below conclusions were drawn post-report by the registrant:

1) At the toxic dose of 71.0 mg AMP/kg bw/day, AMP concentration in dosage form ranged 14.24-14.61 mg/mL (see report p. 124) i.e. 1.42-1.46 % w/w AMP. AMP was tested neutralized to pH 6.6-7.5 using HCl. As is, AMP is alkaline (industrial-grade: pKa = 9.74). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3309 ln(Concentration in % w/w) + 11.548], pH of industrial-grade AMP solutions at 1.42-1.46 % AMP w/w would be 11.7. Turner et al, 2011 (see IUCLID § 4.20) indicate that oral dosage above pH 9 may result in tissue necrosis and vascular thrombosis. Thus, if AMP had been tested as is, the study's toxic dose could not have been reached due to dose-limiting, pH-mediated toxicity. Since OECD guidelines do NOT require any neutralization or pH adjustment, this study represents artificial and non-guideline dose maximization removing AMP's critical toxic effect, which is pH-dependent non-specific toxicity.

2) This is confirmed experimentally by a 13-week oral rat study (Pittz, 1977/79, see IUCLID §7.5.1) done in duplicate at 500 to 1700 mg AMP/kg bw/day with or without neutralisation to pH 6.5-7.3 using HCl. Non-neutralized AMP triggered mortality from 500 mg/kg bw/day due to pH >11 in dosage forms, while neutralized AMP did not cause death up to 1700 mg/kg bw/day. This proves that neutralising AMP artificially increases its maximum tolerated dose (MTD) by a factor of at least 3.5.