Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 2012 - 24 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

EC Number:

279-632-6

EC Name:

Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

Cas Number:

80939-62-4

Molecular formula:

Unspecified

IUPAC Name:

Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: Males: XXg, Females: XXg
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

- Amount of vehicle: 5 mL/kg body weight

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at ≤-70°C until analysis. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
In the Group 1 formulation, no test substance was detected. The concentrations analysed in the formulations of Group 2, 3 and Group 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2, 3 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of Group 2, 3 and Group 4 formulations after storage yielded an absolute relative difference ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 41 - 54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 48 (Group 1), 52, 55, 58 (Group 2), 76 and 77 (Group 4) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 30, 100 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of the dose range finding study where dose levels of 0, 100, 300 and 1000 mg/kg b.w./day were assessed. At 1000 mg/kg b.w./day

Positive control:

no

Examinations

Parental animals: Observations and examinations:

- Mortality / Viability: At least twice daily.
- Clinical signs: Daily, detailed clinical observations were made in all animals
- Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
- Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

Oestrous cyclicity (parental animals):

no

Sperm parameters (parental animals):

From the selected 5 males of the control and high dose group (see Allocation), and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis.

Litter observations:

- Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera (please refer to repeated dose toxicity study)
HISTOPATHOLOGY / ORGAN WEIGHTS
performed on the select animals, please refer to repeated dose toxicity study

Postmortem examinations (offspring):

- Necropsy pups: Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
- Mating index (%) = (Number of females mated / Number of females paired) x 100
- Fertility index (%) = (Number of pregnant females / Number of females paired) x 100
- Conception index (%) = (Number of pregnant females / Number of females mated) x 100
- Gestation index (%) = (Number of females bearing live pups / Number of pregnant females) x 100
- Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

For each group, the following calculations were performed:
- Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation = (Number of dead pups on Day 4 of lactation / Number of live pups at First Litter Check) x 100
- Viability index = (Number of live pups on Day 4 post partum / Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinical signs of toxicity were noted during the observation period

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period. Food consumption before or after allowance for body weight was similar between treated and control animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. For one female at 30 mg/kg b.w./day (no. 68), it was noted that the vaginal smear prepared on Day 4 of the mating period contained sperm cells without a head. This female delivered live offspring with a normal litter size. Spermatogenesis staging revealed no treatment-related changes. Therefore, this finding was considered unrelated to treatment with the test substance.
For female nos. 55, 68 and 73, the number of pups was slightly higher than the number of implantations. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 5 of lactation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Spleen weights were moderately increased in both sexes at 100 mg/kg b.w./day

GROSS PATHOLOGY (PARENTAL ANIMALS)
Enlarged spleen was observed in one male and 4 females at 100 mg/kg b.w./day. Enlarged mesenteric lymph nodes were present in 5 males and all females at 100 mg/kg b.w./day, in 6 females at 30 mg/kg b.w./day and in 2 females at 10 mg/kg b.w./day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related microscopic findings were noted in the mesenteric lymph nodes and adrenal gland cortex (all dose groups). Findings in the mesenteric lymph nodes consisted of:
− foamy macrophages with/without fibrosis (up to a marked degree), which was correlated with enlargement of the mesenteric lymph nodes at necropsy.
− Sinus ectasia (up to a moderate degree) 10, 30 and 100 mg/kg b.w./day, which was considered secondary to the occurrence of foamy macrophages.
− Focal necrosis within the aggregates of foamy macrophages with/without fibrosis of males at 10, 30 and 100 mg/kg b.w./day (up to a moderate degree) and in females at 30 and 100 mg/kg b.w./day (up to a marked degree).
− Macrophage foci up to a moderate degree (females) or marked degree (males) at 10, 30 and 100 mg/kg b.w./day.
Findings in the adrenal cortex consisted of inflammatory lymphocytic cells (up to a slight degree) in females at 10, 30 and 100 mg/kg b.w./day.

HEMATOLOGY (PARENTAL ANIMALS)
slightly increased leucocyte counts in males and females at 100 mg/kg b.w./day, primarily due to increased neutrophil counts. Increased leucocyte counts were also present in females at 30 mg/kg b.w./day, and the percentage of neutrophils was increased in both sexes at this dose.

Effect levels (P0)

1

Dose descriptor:

LOAEL

Remarks:

parental

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg bw/day).

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

DEVELOPMENTAL DATA (OFFSPRING)
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. Gestation index and duration of gestation were normal across the groups. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed. Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings. The statistically significant lower sex ratio at 10 mg/kg b.w./day was considered to be of no toxicological relevance as a dose-related trend was absent.

VIABILITY (OFFSPRING)
One pup of the control group, two pups at 10 mg/kg b.w./day, four pups at 30 mg/kg b.w./day, and one pup at 100 mg/kg b.w./day were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms of pups consisted of scabbing on the snout or in the neck, pale appearance, a blue spot in the neck and a missing tail apex.

BODY WEIGHT (OFFSPRING)
Body weights of pups were unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING)
Incidental macroscopic findings of pups that were found dead included absence of milk in the stomach, autolysis, cannibalism and a missing tail apex. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day).

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No reproduction and developmental toxicity was observed for treatment up to 100 mg/kg b.w./day. Therefore, a reproduction and developmental NOAEL of at least 100 mg/kg b.w./day was derived.