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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate
EC Number:
948-071-5
IUPAC Name:
Reaction mass of C12-14 tert-alkylamines and dimethyl hydrogen phosphate and methyl dihydrogen phosphate
Test material form:
liquid
Specific details on test material used for the study:
-Purity: > 98%
-Physical description: Yellow semi-solid

Test animals

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.corn

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females.Positive evidence
of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed Gestation Day 0 and the
animals were separated. If evidence of copulation was not detected after 14 days of pairing, the animals were separated without further opportunity for mating.
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
Doses / concentrationsopen allclose all
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).

Examinations

Parental animals: Observations and examinations:
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).
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female for 14 days prior to randomization and continuing until evidence of copulation was observed or until termination of the mating period for females with no evidence of mating.
Sperm parameters (parental animals):
Sperm morphology and concentrations were measured. In addition, testes with epididymides and vas deferens were collected for macroscopic examination.
Litter observations:
Each litter was examined daily for survival, and all deaths were recorded. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13. Pups were individually weighed on PND 1, 4, 7, 10, and 13. Pups were individually sexed on PND 0, 4, and 13. On PND 13, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae.
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. 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.
Reproductive indices:
Mating, fertility, and copulation/conception indices were calculated as follows:

Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant)/Total No. of Males (Females) Used for Mating X 100

Male Fertility Index (%) = No. of Males Siring a Litter/Total No. of Males Used for Mating x 100

Male Copulation Index (%) = No. of Males Siring a Litter/ No. of Males with Evidence of Mating(or Females with Confirmed Pregnancy) X 100

Female Fertility Index (%) = No. of Females with Confirmed Pregnancy/Total No. of Females Used for Mating X 100

Female Conception Index (%) = No. of Females with Confirmed Pregnancy/No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100
Offspring viability indices:
Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0/No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)/ No. of Litters Per Group x 100

Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/No. of Litters Per Group x 100
Where N = PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–10, 10–13, and 4 (post-selection)–13

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
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, treatment-related
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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Significantly (p < 0.05) lower mean live litter size on PND 0 was noted in the 250 mg/kg/day group compared to the vehicle control group
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male and female pup birth weights (PND 1) in the 250 mg/kg/day group were lower (29.7% and 29.6%, respectively) than the vehicle control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Male and female F1 pup body weights at 250 mg/kg/day were lower (up to 38.2% and 40.7%, respectively) than the vehicle control group from PND 1 to 13.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on thyroid hormone values in the F1 males and females at any dosage level on PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
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.
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.