Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined screening test (OECD 422): NOAEL = 100 mg/kg bw/day (reproduction)

RA from Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4)

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
(Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Type of information:
experimental study
Adequacy of study:
key 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
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
- 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): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
- Amount of vehicle: 5 mL/kg bw
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 gestation, 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.
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
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 bw/day were assessed. At 1000 mg/kg bw/day
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.
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)
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
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
slightly increased leucocyte counts in males and females at 100 mg/kg bw/day, primarily due to increased neutrophil counts. Increased leucocyte counts were also present in females at 30 mg/kg bw/day, and the percentage of neutrophils was increased in both sexes at this dose level.
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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 bw/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 bw/day (up to a moderate degree) and in females at 30 and 100 mg/kg bw/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 bw/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 bw/day.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: See "Remarks"
Key result
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).
Critical effects observed:
not specified
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
Key result
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).
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
No reproduction and developmental toxicity was observed for treatment up to 100 mg/kg bw/day. Therefore, a reproduction and developmental NOAEL of at least 100 mg/kg bw/day was derived.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Hazard assessment is conducted by means of read-across from a structural analogue/surrogate. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for read-across

There are no data available regarding toxicity to reproduction for Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (CAS 96690-34-5). Therefore, read-across from an appropriate substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-32-4) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII-VIII, 8.4. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in IUCLID Section 13.

CAS 80939-32-4

In a GLP compliant combined 28-days repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422), the source substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4) was given to rats by oral gavage (WIL Reasearch, 2013). Four groups of ten male and ten female Wistar Han rats were exposed to the test substance at 10, 30, or 100 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and thereafter, 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 gestation, and during at least 4 days of lactation. No reproduction toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites). Histopathologically, the assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, and no abnormalities were seen in the reproductive organs of the animal that failed to deliver healthy offspring. Regarding systemic toxicity, treatment with the test substance resulted in effects at all 3 dose levels considered (please refer to chapter 7.5 for details); therefore, the LOAEL was set at the lowest dose level of 10 mg/kg bw/day and thus, no NOAEL could be established. For reproduction  no treatment-related effects occurred and thus, the NOAEL was set at the highest dose level tested, i.e., 100 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Combined screening test (OECD 422): NOAEL = 100 mg/kg bw/day (developmental)

RA fromAmines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4)

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Hazard assessment is conducted by means of read-across from a structural analogue/surrogate. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for read-across

There are no data available regarding genetic toxicity for Amines, C12-14-tert-alkyl, mixed sec-Bu and iso-Bu phosphates (CAS 96690-34-5). Therefore, read-across from an appropriate substance Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-32-4) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII-VIII, 8.4. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

In a GLP compliant combined 28-days repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422), Amines, C11-14-branched alkyl, monohexyl and dihexyl phosphates (CAS 80939-62-4) was given to rats by oral gavage (Key, 2013). Four groups of ten male and ten female Wistar Han rats were exposed to the test substance at 10, 30, or 100 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and thereafter 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 gestation, and during at least 4 days of lactation. No developmental toxicity was observed up to the highest dose level tested (100 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Regarding systemic toxicity, treatment with the test substance resulted in effects at all 3 dose levels considered (please refer to chapter 7.5 for details); therefore, the LOAEL was set at the lowest dose level of 10 mg/kg bw/day and thus, no NOAEL could be established. For developmental toxicity no treatment-related effects occurred and thus, the NOAEL was set at the highest dose level tested, i.e., 100 mg/kg bw/day.

Justification for classification or non-classification

Based on analogy approach, the read across data as reported above provide no indication for reproduction and/or developmental toxicity to be expected from the present substance. Nevertheless, since the data refer to a screening test (OECD 422), these are considered insufficient for any further assessment in terms of classification and labelling.

Additional information