2-amino-2-methylpropanol

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

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

Remarks:

developmental toxicity study: no investigation of repeat-dose toxicity (notably, no information on liver effects) but allows to explore MTD value

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

tested as AMP ULTRA PC 2000 = high-purity grade

Species:

rat

Strain:

other: CRL:CD(SD)

Details on test animals or test system and environmental conditions:

Time-mated female rats were obtained from a commercial supplier. Sexually mature adult rats were 10-11 weeks of age and weighed approximately 200-250 grams at study start.Each animal was evaluated by the laboratory veterinarian to determine the general health status and acceptability for study purposes upon arrival at the laboratory. Rats were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle) and acclimated for at least three days. Prior to dosing animals were acclimated to the wrapping material for approximately 2, 4, and 6 hours on GD 3, 4, and 5, respectively.Animals were housed one per cage in stainless steel cages in rooms designed to maintain adequate environmental conditions for the species. Animals were provided feed and municipal water ad libitum. Rats were stratified by GD 0 body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform mean group weights and standard deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Route of administration:

dermal

Vehicle:

other: deionized water

Details on exposure:

Rats were administered AMP once daily by the dermal (occluded) route at dose levels of 0, 30, 100, or 300 mg/kg/day for approximately 6 hours from GD 6-20. Dose solutions were prepared in deionized water at concentrations of 30, 100, and 300 mg/ml and pH adjusted to approximately 9.5 with HCl. The solutions were administered at a dose volume of 1 ml/kg body weight in order to achieve the targeted dose levels. Dose volumes were adjusted daily based on individual body weights.
Test material was utilized at a pH of approximately 9.5, authors justified as "reflecting the highest pH of most personal care and industrial formulations containing AMP". It should be noted (note from registrant) that ultra-pure undiluted AMP has a much higher pH of 12.2 at 22°C. Since the test item was partially neutralized to prepare dose solutions, the significant parental toxicity related to alcaline properties of AMP is not reflected in this study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose solutions were prepared weekly during the course of the study. Homogeneity and stability were determined prior to study initiation, and concentration of the dose suspensions was determined from the first mix.

Details on mating procedure:

Sexually mature, adult virgin females were naturally mated with males of the same strain at the supplier's facility. Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier, and maintained in the study record. Rats arrived in the testing laboratory on GD 1 or 2.

Duration of treatment / exposure:

GD6-20

Frequency of treatment:

6 hours/day

Duration of test:

15 days

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26 time-mated female rats/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The maternal and developmental toxicity potential of 2-amino-2-methylpropanol (AMP) was evaluated. Groups of 26 time-mated female CD rats were administered AMP in deionized water (pH 9.5) by the dermal route at targeted dose levels of 0, 30, 100, or 300 mg/kg/day from gestation day (GD) 6 through 20. In-life maternal study parameters included clinical observations, body weight, body weight gain, and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and examined for external alterations. Approximately one half of the fetuses were examined for visceral alterations while skeletal examinations were conducted on the remaining fetuses. Blood samples were collected on GD 20 from four presumed pregnant females per group to evaluate blood levels of AMP and confirm its dermal absorption.

Maternal examinations:

A cage-side examination was conducted twice daily, at approximately the same time each day to detect significant clinical abnormalities that were clearly visible upon a limited examination, and used to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Following removal of the wrapping material at the end of each daily six-hour exposure period, the test sites were graded for erythema, edema, scaling, and fissuring. Body weights were recorded on GD 0 by the supplier, and daily during the dosing period, and at necropsy (GD 21). Feed consumption were measured and statistically analyzed for the following intervals: GD 3-6, 6-9, 9-12, 12-15, 15-18, and 18-21. On GD 21, all surviving females (not fasted) were euthanized by carbon dioxide inhalation and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies was counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification. The maternal necropsy included an examination of the external tissues and all orifices. The stomach, liver, and kidneys were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to terminal body weight were calculated. Representative sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of tissues was not conducted. Toxicokinetic SubgroupThe first four presumed pregnant females from each dose group were selected for blood collection to evaluate systemic exposure following dermal administration of AMP. On the last day of dosing (GD20), following approximately three hours of dermal exposure, the designated animals were anesthetized using isoflurane and blood was collected from the orbital sinus. Approximately 0.5 ml of blood from each animal was collected into heparinized tubes. The samples were submitted to the analytical chemistry department for analysis and were stored frozen at approximately -80 C. AMP present in the blood was derivatized with pentafluorobenzoyl chloride under basic conditions and extracted in toluene. Quantitation was performed utilizing an isotopically labeled internal standard (D6-AMP) and matrix standards prepared in control blood. The limit of quantitation was 15 ng/g blood AMP.

Ovaries and uterine content:

A detailed examination of the reproductive tract was performed and the number and position of implantations, viable fetuses, dead fetuses, and resorptions were recorded. Resorptions were classified as either "early" or "late" based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a "dead fetus" would indicate a very recent death as evidenced by a lack external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea were counted. The uteri of females lacking visible implantations were stained with a 10% aqueous solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

Blood sampling:

Toxicokinetic Subgroup: Blood samples were obtained from four presumed pregnant females on the last day of dosing following approximately three hours of dermal application. AMP blood concentration was determined.

Fetal examinations:

The sex of all fetuses was determined and the body weight of all viable fetuses recorded. All fetuses were given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum, and tail. At least one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations. The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder, and reproductive organs. The heads of these fetuses were removed, placed in Bouin's fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue. The remaining fetuses not selected for visceral examination were then skinned, eviscerated, preserved in alcohol and double stained with Alcian Blue and Alizarin Red S for cartilage and bone. After staining, skeletons were macerated and cleared. A thorough evaluation of the fetal skeleton was conducted on the remaining fetuses not selected for visceral examination. All fetal alterations were classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain. The fetal examinations were conducted such that investigators were blind to treatment group assignment.

Statistics:

see below

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Dermal local effects occurred at 300 mg/kg/day: a large majority of animals had slight to severe scaling and fissuring of skin site (slight: 24/26; moderate to severe: 9/26), and scabs (20/26 animals).
Scaling and fissuring also appeared a lower doses, but slight only, and transient, and was considered non adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Toxicokinetic Subgroup
In blood samples taken on the last day following approximately three hours of dermal application, results indicate dermal absorption of AMP in a dose-responsive manner. The disproportionately higher mean blood concentration of AMP at 300 mg/kg/day may have been the result of a compromised skin barrier, as evidenced by significant dermal effects (see above).
Mean AMP concentration in blood was 31, 68 and 732 ng/g at 30, 100 and 300 mg/kg/day, resp, with very high individual variability at the high-dose and a correlation with skin integrity.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

dermal irritation

Remarks on result:

other: local NOAEL

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

dermal irritation

Remarks on result:

other: local LOAEL

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

systemic NOAEL

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

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

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a rat dermal developmental toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, AMP had no effect on development up to 300 mg/kg bw/day.

Executive summary:

In an OECD 414 study, pregnant CD rats were topically administered AMP (adjusted to pH 9.5) at dose levels of 0, 30, 100 or 300 mg/kg bw/day for 6 hours/day over GD 6-20.

• 300 mg/kg bw/day produced local effects: a large majority of animals had slight to severe scaling and fissuring of skin sites (slight: 24/26; moderate to severe: 9/26) and scabs (20/26 animals). There was no maternal systemic or developmental toxicity at any dose level tested.


• 30 and 100 mg/kg bw/day also produced scaling and fissuring, but slight and transient, which was thus considered non adverse.

Under the conditions of this study, the NOAEL for maternal local toxicity was 100 mg/kg bw/day. The NOELs for maternal systemic toxicity and developmental toxicity were both 300 mg/kg bw/day, the highest dose level tested.
Analyses of blood samples confirmed systemic exposure to AMP in a dose-responsive manner and largely supra-proportional systemic exposure at the top-dose due to altered skin barrier. The study was however not designed to quantify percent dermal absorption.

Below conclusions were drawn post-report by the registrant:

1) At the local maternal LOAEL of 300 mg AMP/kg bw/day, AMP concentration in topical solution was 342 mg/mL (see report p. 31) i.e. 34.2%, and solutions were adjusted to pH 9.5. As is, AMP is alkaline (high-purity-grade: pKa = 9.70). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3189 ln(Concentration in % w/w) + 11.401], pH of high-purity-grade AMP solutions at 34.2% AMP w/w would be 12.5. See IUCLID § 4.20: maximum human skin pH is 7 to 8 (Shu-Hua K, 2020) and substances with pH ≥ 11.5 should be considered as Skin Corrosive category 1 and not tested by dermal route (REACH, 2006 amended + ECHA, 2017). Thus, if AMP solutions hadn't been pH-adjusted, the study's maternal LOAEL 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Principles of method if other than guideline:

developmental toxicity study: no investigation of repeat-dose toxicity (notably, no information on liver effects) but allows to explore MTD value

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 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

Details on test material:

The test article was identified in this study as follows:
[14C]-AMP (molecular weight = 91.1 g/mol; specific activity = 25 mCi/mmol)


[14C]-AMP was provided by The Dow Chemical Company prepared in three formulations, identified in this study as follows:

1. AMP (Batch No. TA074801Z1) – Contains 94.85 mg AMP/g and 0.27 mg 14C-AMP/g, for a total calculated value of 95.12 mg AMP/g (Paste)

2. 40% aqueous solution (w/v, pH 9.5) prepared from AMP - Contains 0.26 mg 14C-AMP/g (Liquid)

3. Control Lotion B with pure AMP (Lot BP200500759.06) – Contains 40 mg AMP/g and 0.26 mg 14C-AMP/g, for a total calculated value of 40.26 mg AMP/g (equivalent to the level of AMP in cosmetic lotions)


Pure AMP, 40% aqueous, and Neat Control Lotion B formulations were evaluated for radiochemical concentration, which was determined to be, respectively, 79.3, 86.2, or 87.6% in a first shipment of formulations, and 80.3, 88.9, or 92.4% in a second shipment of formulations.

Specific details on test material used for the study:

tested as AMP ULTRA PC 2000 = high-purity grade

Test animals

Species:

rat

Strain:

other: CRL:CD(SD)

Sex:

female

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

Rats were administered AMP once daily by the dermal (occluded) route at dose levels of 0, 30, 100, or 300 mg/kg/day for approximately 6 hours from GD 6-20. Dose solutions were prepared in deionized water at concentrations of 30, 100, and 300 mg/ml and pH adjusted to approximately 9.5 with HCl. The solutions were administered at a dose volume of 1 ml/kg body weight in order to achieve the targeted dose levels. Dose volumes were adjusted daily based on individual body weights. Test material was utilized at a pH of approximately 9.5, authors justified as "reflecting the highest pH of most personal care and industrial formulations containing AMP".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose solutions were prepared weekly during the course of the study. Homogeneity and stability were determined prior to study initiation, and concentration of the dose suspensions was determined from the first mix.

Duration of treatment / exposure:

15 days: gestation days 6-20

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Dermal local effects occurred at 300 mg/kg/day: a large majority of animals had slight to severe scaling and fissuring of skin site (slight: 24/26; moderate to severe: 9/26), and scabs (20/26 animals). Scaling and fissuring also appeared a lower doses, but slight only, and transient, and was considered non adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

only the reproductive tract was examined

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

other: maximum tolerated dose (MTD)

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Any other information on results incl. tables

for all details, consult the cross-referenced study summary

Applicant's summary and conclusion

Conclusions:

In a rat dermal developmental toxicity study with artificial dose maximization removing AMP's critical toxic effect which is pH-dependent non-specific toxicity, AMP's MTD was >= 300 mg/kg bw/day. No repeat-dose toxicity NOAEL can be determined as the study does not include the necessary examinations (notably, no information on liver effects).

Executive summary:

In an OECD 414 study, pregnant CD rats were topically administered AMP (adjusted to pH 9.5) at dose levels of 0, 30, 100 or 300 mg/kg bw/day for 6 hours/day over GD 6-20.

• 300 mg/kg bw/day produced local effects: a large majority of animals had slight to severe scaling and fissuring of skin sites (slight: 24/26; moderate to severe: 9/26) and scabs (20/26 animals). There was no maternal or developmental toxicity at any dose level tested. 


• 30 and 100 mg/kg bw/day also produced scaling and fissuring, but slight and transient, which was thus considered non adverse. 

Below conclusions were drawn post-report by the registrant:

1) The MTD was >= 300 mg/kg bw/day in females. No repeat-dose toxicity NOAEL can be determined as the study was not designed to include corresponding investigations (notably related to liver effects).

2) At the locally toxic dose of 300 mg AMP/kg bw/day, AMP concentration in topical solution was 342 mg/mL (see report p. 31) i.e. 34.2%, and solutions were adjusted to pH 9.5. As is, AMP is alkaline (high-purity-grade: pKa = 9.70). Based on Fernandes, 2023 [see IUCLID § 4.20: pH = 0.3189 ln(Concentration in % w/w) + 11.401], pH of high-purity-grade AMP solutions at 34.2% AMP w/w would be 12.5. See IUCLID § 4.20: maximum human skin pH is 7 to 8 (Shu-Hua K, 2020) and substances with pH ≥ 11.5 should be considered as Skin Corrosive category 1 and not tested by dermal route (REACH, 2006 amended + ECHA, 2017). Thus, if AMP solutions hadn't been pH-adjusted, the study's LOAEL 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.

3) 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.