Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate

Endpoint summary

Description of key information

The objectives of the study were to evaluate the potential toxic effects of the test material when administered to rats for at least 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through day 13 of postnatal life. The study was performed to the standardized guideline OECD 422, under GLP conditions.

 

Based on these results, the NOAEL for parental systemic toxicity was considered to be 75 mg/kg/day.

 

The NOAEL for neonatal toxicity was 75 mg/kg/day based on the lower live litter size, lower postnatal survival, and lower pup body weights and body weight gains in the 250 mg/kg/day group (Charles River, 2017).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2017 to 02 November 2017

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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

-Purity: > 98%
-Physical description: Yellow semi-solid

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Following receipt and until pairing, all F0 animals were housed 2–3 rats/cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH).

The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River.

Actual mean daily temperature ranged from 72.5°F to 73.0°F (22.5°C to 22.8°C) and mean daily relative humidity ranged from 38.4% to 56.7% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod.

Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The vehicle was dispensed approximately weekly for administration to the vehicle control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the vehicle control group and stored refrigerated (2°C to 8°C), protected from light. The vehicle was mixed throughout the sampling and dose administration procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for concentration analysis were collected from the middle stratum of first, middle, and last dosing formulations (including the vehicle control group) prepared that included all dose groups. One set of samples from each collection was analyzed using a validated high performance liquid chromatography method with charged aerosol detection. The retention time of the test substance was approximately 1.5 to 2.1 minutes.

Duration of treatment / exposure:

Males and females were approximately 10 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49–54 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postcohabitation Day 25 or Postmating Day 25, respectively) for a total of 40–52 doses.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The dose selection was based upon a range-finding study (please see RSS section 7.5.1 Supporting study, Charles River, 2017 (range-finding)).

Observations and examinations performed and frequency:

All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

Clinical observations, body weights, and food consumption recorded at appropriate intervals.

Motor activity was assessed for 5 animals/sex/group during the last week of dose administration (Study Day 25, males) or on Lactation Day 13 (females).

Blood samples for clinical pathology evaluations (hematology, coagulation, and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (Study Day 28 for males and Lactation Day 14 for females).

Sacrifice and pathology:

A complete necropsy was conducted on all F0 parental animals found dead or at the scheduled termination.

All F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Females and males in the 250 mg/kg/day group had clinical observations of tremors, clonic convulsions, rales, and/or dilated pupils noted primarily at approximately 1.5 hours following dose administration; the observations of tremors, clonic convulsions, and dilated pupils were noted generally 1–4 days prior to the day of death while rales was observed throughout.

Other remarkable clinical observations noted occasionally throughout the dosing period for females that were found dead at 250 mg/kg/day included yellow material on various body surfaces (hindlimbs, urogenital area, anogenital area, and/or ventral trunk) noted at the detailed physical and/or daily examinations, and salivation, clear and/or red material around the mouth and/or nose, and clear nasal discharge noted at approximately 1.5 hours following dose administration.

Mortality:

mortality observed, treatment-related

Description (incidence):

5 females were found dead at 250 mg/kg/day. Histologic findings indicate acute inflammation in the non-glandular stomach.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, significantly (p < 0.05 or p < 0.01) lower mean body weight gains were noted in the 250 mg/kg/day group F0 males during Study Days 0–7 and 13–21 compared to the vehicle control group; mean body weight gains in this group were similar to the vehicle control group during Study Days 7–13 and 21–27.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower mean food consumption was noted in the 250 mg/kg/day group F0 males during Study Days 0–7 and 7–13 compared to the vehicle control group; the difference was significant (p < 0.05) during Study Days 0–7.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A significantly (p < 0.05) lower activated partial thromboplastin time (APTT) value was noted in the 250 mg/kg/day group F0 males compared to the vehicle control group; this difference was of minimal magnitude and was considered to be the result of normal biological variation and not test substance-related.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A significantly (p < 0.05) higher mean adrenal gland weight relative to final body weight was noted in the 250 mg/kg/day group F0 males. There were no histologic correlates and the mean remained within the historical control reference range. Thus, this finding was considered to be due to biologic variation and not test substance-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related raised and eroded areas in the stomach were noted in the 250 mg/kg/day group F0 females. These observations correlated with acute inflammation in the non-glandular stomach.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the kidneys and stomach (glandular and non-glandular), these alterations were not considered adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males at any dosage level.

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Critical effects observed:

no

Conclusions:

The NOAEL for systemic toxicity was considered to be 75 mg/kg/day.

Executive summary:

The objectives of the study were to evaluate the potential toxic effects of the test material when administered to rats for at least 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through day 13 of postnatal life. The study was performed to the standardized guideline OECD 422, under GLP conditions.

 

Under the conditions of this screening study, no test substance-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 250 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test material when administered orally by gavage to Crl:CD(SD) rats.

 

Adverse lower mean body weights, body weight gains, and food consumption were noted for F0 males throughout the study and for F0 females at the beginning of the premating period and during gestation in the 250 mg/kg/day group.

 

Macroscopic findings consisted of raised and eroded areas in the stomach noted in the 250 mg/kg/day group females and histologic findings consisted of inflammation in the glandular and non-glandular stomach noted in the 250 mg/kg/day group males and females.

 

Based on these results, the NOAEL for parental systemic toxicity was considered to be 75 mg/kg/day.

 

The NOAEL for neonatal toxicity was 75 mg/kg/day based on the lower live litter size, lower postnatal survival, and lower pup body weights and body weight gains in the 250 mg/kg/day group.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat