Registration Dossier
Registration Dossier
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EC number: 271-668-0 | CAS number: 68603-62-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
NOEL & NOAEL (male fertility) = 500 mg/kg bw/d); OECD 422; Barraclough, 2018
NOEL & NOAEL (female fertility) = 100 mg/kg bw/d); OECD 422; Barraclough, 2018
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 July 2017 to 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to same 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 (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- other: CRL:WI(Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:
Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported, assume yes
- Age at study initiation:
10-11 weeks old
- Weight at study initiation:
Males: 288.6 - 391.8 g; females: 173.2 - 235.3 g
- Fasting period before study:
No
- Housing:
During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum):
Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum):
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
- Acclimation period:
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 days prior to initiation of smearing (females).
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 ºC
- Humidity (%):
30-70 % RH
- Air changes (per hr):
15-20
- Photoperiod (hrs dark / hrs light):
12:12
IN-LIFE DATES: From: 26 July 2017 To: 25 September 2017 - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- - PREPARATION OF DOSING SOLUTIONS:
##
- VEHICLE
- Justification for use and choice of vehicle (if other than water):
Homogenous stable solutions were prepared in corn oil, a guideline recommended vehicle.
- Concentration in vehicle:
##
- Amount of vehicle (if gavage):
5 mL/kg
- Lot/batch no. (if required):
MKBP7039V, MKBZ9899V and MKCC0462
- Purity: Assumed pure corn oil - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: Up to 15 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 (GD 0) of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for up to an additional 5 days.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Females were housed individually during gestation, and with their litter during the lactation phase.
- Any other deviations from standard protocol: No - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Single 5.0 mL aliquot formulation samples were collected from the main in-life experiment study for each animal group at the respective concentrations (0, 20.0, 60.0 and 100.0 mg/mL) on the 26th July 2017 (week 1 of the in-life phase), 9th August 2017 (week 3 of the in-life phase) and 30th August 2017 (week 6 of the in-life phase) for analysis.
A validated gradient elution reversed-phase high-performance liquid chromatographic method with ultraviolet detection (HPLC-UV) was employed for determination of formulation stability and homogeneity, and concentration analyses of formulations administered during the study.
No test item was observed in the control group samples. Measured concentrations from the test item groups were found to be in the range of 89.2 - 102.3 % of nominal values. - Duration of treatment / exposure:
- Males: 42 consecutive days (2 weeks prior to pairing; during pairing; and until the day before necropsy)
Females: 61 days (2 weeks prior to pairing [pre-pairing phase]; during the pairing phase; and until LD 13, inclusive, or 25 days post-coitum for females which did not litter and were sent to necropsy on LD 14 or 26 days post coitum). Some females were not dosed on LD 0 if they were observed to be starting or just completed parturition. - Frequency of treatment:
- Daily
- Details on study schedule:
- N/A
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10 males and 10 females per treatment group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses selected based on the results of a preliminary range-finder test.
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A - Positive control:
- N/A
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
BODY WEIGHT: Yes
- Time schedule: Male body weights were recorded once during acclimation, before dosing on the first day of dosing, at weekly intervals, and before necropsy. Female body weights were recorded once during acclimation; before dosing on the first day of dosing; at weekly intervals prior to pairing and until confirmation of mating; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 1, 4, 13 and 14 (prior to necropsy).
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 0 to 13. Consumption was calculated as g/animal/day.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The amount of water consumed was determined daily during the pre-pairing phase (both sexes). Consumption was calculated as g/animal/day.
OTHER: N/A - Oestrous cyclicity (parental animals):
- Daily vaginal washings were conducted for all females during acclimation (predose) from 1 week after arrival until the day prior to dosing. The stage of estrous was recorded, and only females with regular 4 to 5 day cycles were included on study.
Daily vaginal washings were conducted on females from the start of dosing until the confirmation of mating and on LD 14, prior to necropsy. - Sperm parameters (parental animals):
- Parameters examined in F0 male parental generations:
testis weight, epididymis weight - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed, sexed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups
GROSS EXAMINATION OF DEAD PUPS:
After sacrifice, surplus pups culled on PND 4 had the sex determined; no macroscopic examination was required.
After sacrifice, decedent pups and one pup/sex/litter sacrificed on PND 13 were examined internally. The remaining pups sacrificed were examined externally for gross abnormalities, with particular attention to the external reproductive genitals.
Thyroid glands of pups culled on PND 13 (one pup/sex/litter across groups) were weighed approximately 24 hours post-fixation, and then and preserved in 10% neutral buffered formalin
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: N/A
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: N/A - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals on post-pairing Day 14 after the preliminary evaluation of female data and an overnight period without food.
- Maternal animals: All surviving animals on lactation Day (LD) 14 (those that achieved pregnancy) or Day 26 post coitum (those that did not litter) after an overnight period without food.
GROSS NECROPSY
- Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 3 were prepared for microscopic examination and weighed, respectively. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring were sacrificed at 13 days of age (study terminus) or at 4 days of age (litter standardisation).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Pups sacrificed were examined externally for gross abnormalities, with particular attention to the external reproductive genitals.
To provide one pooled sample for each litter, where possible, pups culled on PND 4 had blood samples (1 x 0.6 mL) withdrawn for thyroid hormone analysis via decapitation.
To provide two pooled samples for each litter from Groups 1, 2, and 3 (one sample from two males and one sample from two females, where possible), pups sacrificed on PND 13 had blood samples (2 x 0.8 mL) withdrawn for thyroid hormone analysis by cardiac puncture. All pups sacrificed on PND 13 had blood samples (1 x 0.8 mL) withdrawn for thyroid hormone analysis by cardiac puncture.
GROSS NECROPSY
- After sacrifice, decedent pups and one pup/sex/litter sacrificed on PND 13 were examined internally. The remaining pups sacrificed were examined externally for gross abnormalities, with particular attention to the external reproductive genitals.
HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands of pups culled on PND 13 (one pup/sex/litter across groups) were weighed approximately 24 hours post-fixation, and then and preserved in 10% neutral buffered formalin. - Statistics:
- Data for each sex were analyzed separately, unless stated otherwise. Only data collected on or after the first day of dosing were analyzed statistically. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P ≤ 0.05. By default, significant results were reported as *P ≤ 0.05, +P ≤ 0.01, and/or #P ≤ 0.001.
- Reproductive indices:
- Male and female mating
Fecundity
Fertility indices - Offspring viability indices:
- Duration of gestation
Number of implantation sites
Number of pups born
Number of pups alive on PND 1, 4 (before culling)
% male pups on PND 1
Percent post-implantation loss
Live birth and survival indices PND 1-4
Pup weights
Ano-genital distance (males only)
Thyroid hormone - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Urogenital staining and wet abdominal fur were occasionally recorded during Pre Pairing and Pairing for one or two males administered 300 or 500 mg/kg/day. An observation of vocalizing on handling was noted in one female from each test article treated group from Pre-Pairing Day 4 or 5. One female administered 500 mg/kg/day was noted to have wet fur of the anogenital area and/or abdomen during the pre-pairing phase or GD 7. These observations were attributed to test article.
Incidences of mouth rubbing were noted immediately upon the return to the home cage in up to 4 of 10 females administered 500 mg/kg/day. Salivation was observed from Pre-Pairing Day 8 for some females administered 100, 300, or 500 mg/kg/day and persisted through pairing for occasional females, but no dose response was noted. Salivation was not recorded during gestation or lactation. Salivation was also noted for males administered 100, 300, or 500 mg/kg/day, without a dose response, from Pre-Pairing Day 9, 7, and 10, respectively, and until the end of the pre-pairing, through the pairing, and into the post pairing phases. These observations were attributed to the consistency and palatability of the test article and were considered non-adverse.
All other observations, including fur staining, fur loss, sores/lesions, and tail damage, were considered incidental and not related to the test article as they were also observed in control animals, showed no dose relationship, and/or are commonly observed in animals of this age and strain. - Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality:
- not specified
- Description (incidence):
- One male administered 500 mg/kg/day was found dead on Study Day 16 (Pairing Day 1). No adverse clinical observation were noted prior to the animal being found dead, and no specific cause of demise could be determined from the tissues examined. The death, therefore, had an uncertain relationship to the test article.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article-related lower mean body weight gain was recorded for males administered 300 or 500 mg/kg/day. Mean male body weight gain was statistically significantly reduced during the pre pairing phase for males administered 500 mg/kg/day and was 67% lower than controls by the end of the pre-pairing phase. Thereafter, mean body weight gain for males was similar to or slightly lower than controls, but this did not attain statistical significance. Overall, the mean male body weight gain at completion of the study was statistically significantly reduced at 57% of the control value for males administered 500 mg/kg/day. While individual incidences did not attain statistical significance for males administered 300 mg/kg/day, the overall gain for the study showed a statistically significantly reduction (77% of the control value). Mean body weight was unaffected in males administered 100 mg/kg/day, compared with controls.
Body weight gain was also lower for females administered 500 mg/kg/day, when compared with controls during late gestation.
Mean body weight during the pre-pairing, gestation, or lactation phases for females administered 100, 300 mg/kg/day were compared with controls and considered unaffected by the test article. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Lower food consumption was noted for males and females administrated 500 mg/kg/day during the first two weeks of dosing. Food consumption was also noted to be lower for 500 mg/kg/day females during late gestation, when compared with controls.
No effect on food consumption was noted following 100 or 300 mg/kg/day administration. - Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- A test article related effect on water consumption was noted for animals administered 300 or 500 mg/kg/day.
Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, compared with controls, such that at the end of the pre-pairing phase, male water consumption was 67 or 100% greater than controls, respectively, and female water consumption was 38 or 80% greater than controls, respectively. - Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Hematology parameters were unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- A statistically significant elevation in glucose levels was noted in males administered 500 mg/kg/day, and a statistically significant increase in cholesterol, calcium, and inorganic phosphate levels was noted in males administered 300 or 500 mg/kg/day, compared with control. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day.
A statistically significant increase in TSH was noted in males administered 300 mg/kg/day (155 % of control) and animals administered 500 mg/kg/day (188 and 176 % of control) following approximately 6 weeks of dosing. These values fell outside of background control ranges. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean urine volume was increased for males administered 300 or 500 mg/kg/day, compared with controls (121 and 86 % of control values, respectively); the increase attained statistical significance for males administered 300 mg/kg/day.
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- During the assessments, moderate salivation, piloerection, and changes in body/head posture were observed for males in all test article treated groups, compared with controls. In addition, a dose related increase in ataxia was noted, and high stepping, walking on toes, and slow deliberate movements were noted for animals administered 300 or 500 mg/kg/day, although a decrease in activity was not observed in these groups, compared with controls. A statistically significant increased number of rears were noted on Pre-Pairing Day 6 for males administered 300 or 500 mg/kg/day and Post-Pairing Day 12 for males administered 500 mg/kg/day, compared with controls.
Female changes during the assessments included piloerection and salivation, which were generally dose related and behavior changes predominantly characterized by paw flicking (fore paws). A statistically significant decreased number of rears were noted on LD 1 and 7 for females 300 mg/kg/day, compared with controls. A lower number was also recorded on LD 1 for females administered 500 mg/kg/day; these females were early decedents due to litter loss and, therefore, no data are available for LD 7. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Locomotor activity was unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- A range of Amines-related findings were recorded in the liver, thyroid, kidney, and nonglandular stomach of animals administered 100, 300, or 500 mg/kg/day.
In the liver, centrilobular hepatocyte hypertrophy was recorded in two males administered 100 mg/kg/day, all males and all examined females administered 300 or 500 mg/kg/day. This finding was characterized by the enlargement of the hepatocytes, a ground glass appearance of the cytoplasm, and slight compression of the adjacent sinusoids; was principally localized within the centrilobular areas, but occasionally also extended more diffusely to midzonal and periportal regions in affected animals; and correlated generally with increased group mean adjusted liver weights and the macroscopic finding of dark/mottled discoloration.
In the thyroid, follicular cell hypertrophy was recorded in one male administered 100 mg/kg/day, most males and four females administered 300 mg/kg/day and all males and one female administered 500 mg/kg/day and was characterized by follicular cells with increased amounts of cytoplasm, follicular epithelium that ranged from flat to cuboidal or columnar and follicles with decreased amounts of colloid. This finding correlated generally with increased group mean adjusted thyroid weights and with the macroscopic finding of dark discoloration in one male administered 500 mg/kg/day.
In the kidney, nephropathy was recorded in all examined males administered Amines, which was characterized by multifocal areas of tubular basophilia, inflammatory foci dominated by lymphocytes and/or plasma cells, and granular casts principally located at the corticomedullary junction, but occasionally also in cortical and/or papillary tubules. In addition, hyaline droplets in the cytoplasm of tubular epithelial cells of males administered 100, 300 or 500 mg/kg/day were recorded with higher gradings, compared with concurrent controls. This finding was characterized by focally extensive areas of brightly eosinophilic aggregates of cytoplasmic droplets principally located in the superficial cortex, but multifocally extending towards to the corticomedullary junction. Both of these microscopic findings correlated generally with increased group mean adjusted kidney weights and with the macroscopic finding of mottled and/or pale discoloration.
In addition, in the nonglandular stomach, epithelial hyperplasia and orthokeratotic hyperkeratosis were recorded in one male administered 300 mg/kg/day and four males administered 500 mg/kg/day; these correlated generally with mucosal thickening or the presence of a raised focus, macroscopically - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- No adverse effects on the estrous cycles were observed following test article administration.
An increased length of estrous cycles was recorded for females administered 500 mg/kg/day in pre-pairing. However, as the pre-pairing mean cycle length was reduced in females administered 300 mg/kg/day, the mean number of estrous cycle lengths was unaffected values were within historical background control ranges this observation is considered to be incidental. - Reproductive function: sperm measures:
- not examined
- Description (incidence and severity):
- N/A
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Mating and fertility were unaffected by test article administration.
The majority of animals mated within the first four days of pairing. Two females administered 300 mg/kg/day and two females administered 500 mg/kg/day did not mate within one estrus (4/5 days). However, mating did occur and the females achieved pregnancy, therefore, the delay in mating was considered to be of little toxicological significance.
One control female and one female administered 300 mg/kg/day were sent to necropsy on GD 26 having failed to produce a litter. At necropsy, the animals were found to be non-pregnant. The non-pregnant finding for these animals was considered incidental, and no relationship to dose was noted. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- reproductive performance
- Key result
- Critical effects observed:
- no
- Clinical signs:
- not examined
- Description (incidence and severity):
- N/A
- Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality:
- not examined
- Description (incidence):
- N/A
- Body weight and weight changes:
- not examined
- Description (incidence and severity):
- N/A
- Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- N/A
- Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- not examined
- Description (incidence and severity):
- N/A
- Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- not examined
- Description (incidence and severity):
- N/A
- Clinical biochemistry findings:
- not examined
- Description (incidence and severity):
- N/A
- Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Behaviour (functional findings):
- not examined
- Description (incidence and severity):
- N/A
- Immunological findings:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- not examined
- Description (incidence and severity):
- N/A
- Gross pathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Neuropathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings: non-neoplastic:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings: neoplastic:
- not examined
- Description (incidence and severity):
- N/A
- Other effects:
- not examined
- Description (incidence and severity):
- N/A
- Details on results:
- N/A
- Reproductive function: oestrous cycle:
- not examined
- Description (incidence and severity):
- N/A
- Reproductive function: sperm measures:
- not examined
- Description (incidence and severity):
- N/A
- Reproductive performance:
- not examined
- Description (incidence and severity):
- N/A
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Excluding the total litter death noted for the litters of dams administered 500 mg/kg/day, no other test article-related pup clinical observations were noted.
- Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- A test article-related effect on live birth index for dams administered 500 or 300 mg/kg/day and pup survival for litters of dams administered 500 mg/kg/day was adversely affected.
Nine of 10 females administered 500 mg/kg/day produced a litter. Five litters were recorded to have live pups at birth, with a single litter surviving to scheduled necropsy; in this litter only 4 of 12 pups alive on PND 1. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. One litter contained still born pups only, and, excluding one litter which survived to PND 13, all others had total litter death by PND 2.
An effect was observed for the live birth index of females administered 300 mg/kg/day compared with controls (83.4% compared to 100% for control), but postnatal survival was comparable with control values.
One litter from a control dam and one litter from a dam administered 300 mg/kg/day died by PND 2. Another animal had 12 pups in total, two still born pups and all remaining pups found dead with no milk evident in the stomach on PND 0. A third animal had 1 pup in total, which was noted to be cold to touch, weak, unfed, and was found dead on PND 2. The deaths were considered incidental as they were noted in controls or occurred as a single incident in the group, with no other effects on pup survival. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A test article-related effect on body weight and body weight gain was noted for pups from litters of dams administered 300 or 500 mg/kg/day, compared with controls.
Only two litters survived to PND 1 and only one litter survived to PND 13 due to the number of total litter losses between PND 0 and 2 observed for litters of dams administered 500 mg/kg/day. Of the few surviving litters, a reduction in body weight was noted, compared with controls. Where statistical evaluation was possible (PND 1), this reduction attained a statistical significance.
A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in males, females, and/or combined values on all body weight occasions. A reduction in combined body weight gain of 2.9 g was also noted between PND 1 to 13.
No test article-related effect was noted in litters of dams administered 100 mg/kg/day. - Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- N/A
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- not examined
- Description (incidence and severity):
- N/A
- Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- not examined
- Description (incidence and severity):
- N/A
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No effect of test article on pup thyroid hormone levels was noted. All values, including the values that attained statistical significance, were considered incidental and not related to the test article as no dose relationship was apparent and/or the values were within background ranges.
- Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Sexual maturation:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- not examined
- Description (incidence and severity):
- N/A
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Compared with concurrent controls, no treatment-related changes to pup thyroid weights on PND 13 were suggestive of effects due to test article administration to the adults.
- Histopathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Other effects:
- not examined
- Behaviour (functional findings):
- not examined
- Description (incidence and severity):
- N/A
- Developmental immunotoxicity:
- not examined
- Description (incidence and severity):
- N/A
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- mortality
- body weight and weight gain
- Key result
- Critical effects observed:
- no
- Clinical signs:
- not examined
- Description (incidence and severity):
- N/A
- Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality / viability:
- not examined
- Description (incidence and severity):
- N/A
- Body weight and weight changes:
- not examined
- Description (incidence and severity):
- N/A
- Food consumption and compound intake (if feeding study):
- not examined
- Description (incidence and severity):
- N/A
- Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Water consumption and compound intake (if drinking water study):
- not examined
- Description (incidence and severity):
- N/A
- Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- not examined
- Description (incidence and severity):
- N/A
- Clinical biochemistry findings:
- not examined
- Description (incidence and severity):
- N/A
- Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Sexual maturation:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- not examined
- Description (incidence and severity):
- N/A
- Gross pathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Histopathological findings:
- not examined
- Description (incidence and severity):
- N/A
- Other effects:
- not examined
- Description (incidence and severity):
- N/A
- Behaviour (functional findings):
- not examined
- Description (incidence and severity):
- N/A
- Developmental immunotoxicity:
- not examined
- Description (incidence and severity):
- N/A
- Key result
- Reproductive effects observed:
- yes
- Lowest effective dose / conc.:
- 300 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to other toxic effects:
- reproductive effects as a secondary non-specific consequence of other toxic effects
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Conclusions:
- Males - Reproductive performance was not affected by the test article.
Females - Reproductive performance was not affected by the test article
Parturition and litter - Test article-related effects on litter survival were noted. Eight of 10 females administered 500 mg/kg/day were sent to necropsy between LD 0 and 2 due to total litter loss. In all females that successfully littered, up to 100% of pups were still born, with three litters which contained still born pups only. The litter death was attributed to the test article and was adverse. - Executive summary:
OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.
A summary of adult reproductive responses to the test item are described below;
Males
Reproductive performance was not affected by the test article i.e. no test-article related effects on mating or fertility were noted.
Females and litter
Nine of 10 females administered 500 mg/kg/day produced a litter, of which one entire litter was stillborn. For females administered 500 mg/kg/day, between 22 to 100 % of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. Eight litters were recorded with live pups at birth, with total litter losses noted by PND 2 for four of these litters, with only one litter surviving to scheduled necropsy on PND 13.
The litter death was attributed to the test article and was considered adverse.
A test article-related effect on the live birth index was observed following 300 mg/kg/day administration (83.4% compared to 100% for controls), however, post-implantation survival index and pup survival indices were comparable with controls and sex ratio was unaffected
Endpoint discussion
Test article-related findings observed in males following 500 mg/kg/day administration were generally confined to systemic toxicity in the rat. These findings were considered not to represent an adverse effect in reproduction, and hence, the no observed effect level (NOEL) for reproductive toxicity in males was established as 500 mg/kg/day. The NOEL for females reproductive toxicity was established as 100 mg/kg/day based on the number of litter losses observed in the 500 mg/kg/day test group and
the test article-related effect on the live birth index following 300 mg/kg/day administration.
Reference
Mean number of implantation sites was unaffected by test article administration, however, post-implantation survival index was lower in the 500 mg/kg/day dose group, compared with controls, resulting from a total in utero litter loss for one female from this group.
Live birth and pup survival (PND 1-4) indices were adversely affected following 500 mg/kg/day administration. Nine of 10 females from this dose group produced a litter, of which one entire litter was stillborn. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. Eight litters were recorded with live pups at birth, with total litter losses noted by PND 2 for four of these litters, with only one litter surviving to scheduled necropsy on PND 13. From the surviving litter, only 4 of 12 pups were alive on PND 1. Due to the survival of only one litter, assessment of sex ratio (% males) could not be accurately assessed for this dose level.
A test article-related effect on the live birth index was observed following 300 mg/kg/day administration (83.4% compared to 100% for controls), however, post-implantation survival index and pup survival indices were comparable with controls and sex ratio was unaffected.
One 300 mg/kg/day litter and one control litter died by PND 2. Another 300 mg/kg/day female had 12 pups in total, two still born pups and all remaining pups found dead with no milk evident in the stomach on PND 0. Another 300 mg/kg/day female had 1 pup in total, which was noted to be cold to touch, weak, unfed, and was found dead on PND 2. The deaths were considered incidental as they were noted in controls or occurred as a single incident in the group, with no other effects on pup survival.
No test article-related effects were noted in litters following maternal administration of 100 mg/kg/day.
Table 4 Group mean achieved dose rates for each treatment group (in mg/kg bw/day)
Test Group (mg/kg/day) |
Nominal formulation concentration (mg/mL) |
Actual formulation concentration achieved (% of nominal) |
||
Week 1 |
Week 3 |
Week 6 |
||
Control |
0 |
nd |
nd |
nd |
100 |
20 |
95.8 |
97.3 |
102.3 |
300 |
60 |
96.9 |
95.7 |
96.3 |
500 |
100 |
94.8 |
95.9 |
89.2 |
nd: not detected
Table 5 Mean body weights and body weight gains/ losses (n=10 unless stipulated otherwise) (F0)
Sex / Day |
Bodyweight (g) |
|||
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
|
MALES |
||||
Predose |
||||
Day 1 |
301.5 |
317.7 |
313.0 |
305.7 |
Pre-pairing |
||||
Day 1 |
325.1 |
342.6 |
339.0 |
328.4 |
Day 8 |
345.1 |
365.8 |
355.2 |
331.6 |
Day 15 |
363.1 |
382.3 |
370.8 |
341.0 |
Change Day 1-8 |
20.1 |
23.2 |
16.2 |
3.3** |
Change Day 8-15 |
18.0 |
16.5 |
15.5 |
9.3* |
Pairing |
||||
Day 7 |
369.2 |
389.2 |
372.7 |
348.6 (n=9) |
Day 14 |
385.1 |
406.3 |
385.5 |
365.1 (n=9) |
Change Day 7-14 |
15.9 |
17.1 |
12.8 |
16.5 |
Post-pairing |
||||
Day 7 |
400.6 |
420.8 |
396.2 |
374.9 (n=9) |
Day 13 |
407.5 |
424.2 |
402.2 |
378.2 (n=9) |
Change Day 7-13 |
6.8 |
3.4 |
6.0 |
3.3 |
Overall weight change (pre-pairing Day 1 – post-pairing Day 13) |
82.5 |
81.6 |
63.2* |
47.0** |
FEMALES |
||||
Predose |
||||
Day 8 |
190.1 |
189.6 |
208.6 |
217.3 |
Pre-pairing |
||||
Day 1 |
198.1 |
197.9 |
208.6 |
215.2 |
Day 8 |
208.6 |
199.4 |
212.1 |
225.4 |
Day 15 |
217.3 |
200.6 |
209.4 |
219.4 |
Change Day 1-8 |
10.5 |
10.8 |
12.8 |
8.8 |
Change Day 8-15 |
8.6 |
6.5 |
13.3 |
10.0 |
Gestation |
||||
Day 0 |
220.6 (n=9) |
242.0 |
268.4 (n=9) |
326.9 (n=9) |
Day 7 |
217.5 (n=9) |
238.8 |
263.4 (n=9) |
314.7 (n=9) |
Day 14 |
228.0 (n=9) |
247.3 |
277.1 (n=9) |
332.3 (n=9) |
Day 20 |
221.0 (n=9) |
244.6 |
269.1 (n=9) |
311.6 (n=9) |
Change Day 0-7 |
21.4 |
21.3 |
19.4 |
23.7 |
Change Day 7-14 |
26.4 |
24.5 |
29.8 |
24.4 |
Change Day 14-20 |
58.5 |
51.3 |
55.2 |
42.6 |
Lactation |
||||
Day 1 |
252.8 (n=9) |
252.3 |
255.3 (n=8) |
- |
Day 4 |
256.6 (n=8) |
259.4 |
261.6 (n=8) |
- |
Day 13 |
275.5 (n=8) |
274.9 |
286.8 (n=8) |
- |
Day 14 |
258.2 (n=8) |
255.0 |
254.9 (n=8) |
- |
Change Day 1-4 |
3.7 |
7.1 |
6.4 |
- |
Change Day 4-13 |
18.9 |
15.6 |
25.2 |
- |
Change Day 13-14 |
-17.3 |
-20.0 |
-31.9* |
- |
Overall weight change (pre-pairing Day 1 – gestation Day 20) |
128.6 |
116.8 |
134.0 |
110.7 |
* p<0.05, **p<0.01
Table 6 Haematology, clinical chemistry, urinalysis and pathology findings (F0)
Doses (ppm) |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Male |
Female |
|||||||
Pathology (remarkable changes) |
||||||||
Adrenals - pale, moderate - large, slight - pale, slight |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 1 0 0 |
(n=5) 0 1 1 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=1) 0 0 0 |
Thyroid - dark, slight - pale, slight - small, slight |
(n=10) 0 0 0 |
(n=10) 0 0 0 |
(n=10) 0 0 0 |
(n=9) 1 0 0 |
(n=8) 0 1 1 |
(n=10) 0 0 0 |
(n=8) 0 2 1 |
(n=1) 0 0 0 |
Thymus - dark, minimal - red focus - small, slight |
(n=5) 0 0 0 |
(n=5) 1 0 0 |
(n=5) 0 1 0 |
(n=5) 0 1 0 |
(n=5) 0 0 0 |
(n=5) 0 0 0 |
(n=5) 0 0 1 |
(n=1) 0 0 0 |
Lymph node, mesenteric - red, slight |
(n=5) 0 |
(n=5) 2 |
(n=5) 0 |
(n=5) 1 |
(n=5) 1 |
(n=5) 1 |
(n=5) 1 |
(n=1) 0 |
Liver - mottled, moderate - mottled, slight - pale, moderate - pale, slight |
(n=7) 1 1 0 2 |
(n=5) 0 4 0 1 |
(n=7) 0 3 1 2 |
(n=7) 1 3 0 2 |
(n=5) 0 0 0 0 |
(n=5) 0 0 0 0 |
(n=5) 0 0 0 0 |
(n=1) 0 0 0 0 |
Stomach - raised focus, pale - red, slight - striation, red - thick, minimal - thick, slight |
(n=5) 0 1 0 0 0 |
(n=5) 0 1 0 0 0 |
(n=5) 0 1 2 1 1 |
(n=5) 1 0 2 0 1 |
(n=5) 0 0 0 0 0 |
(n=5) 0 1 0 0 0 |
(n=5) 0 0 0 0 0 |
(n=1) 0 0 0 0 0 |
Colon - distension, slight |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Lymph node, mandibular - red, moderate |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Kidney - mottled, minimal - mottled, moderate - mottled, slight - pale, moderate - pale, slight - pelvic dilation, minimal - pelvic dilation, slight - red area |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 2 0 2 0 0 1 |
(n=8) 0 1 5 1 0 1 1 0 |
(n=7) 1 1 2 1 0 0 0 0 |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 0 0 0 0 0 0 |
(n=5) 0 0 0 0 0 0 1 0 |
(n=5) 0 0 0 0 0 0 0 0 |
Testis - small, severe |
(n=10) 0 |
(n=10) 1 |
(n=10) 0 |
(n=9) 0 |
n/a |
n/a |
n/a |
n/a |
Epididymis - small, marked |
(n=10) 0 |
(n=10) 1 |
(n=10) 0 |
(n=9) 0 |
n/a |
n/a |
n/a |
n/a |
Skin - fur loss, moderate - fur loss, slight - sore, slight |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=0) 0 0 0 |
(n=2) 1 0 1 |
(n=2) 0 2 1 |
(n=0) 0 0 0 |
Lung - pale focus |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=1) 0 |
Tail - kinked |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
(n=2) 2 |
(n=0) 0 |
(n=0) 0 |
(n=0) 0 |
Peyer’s Patch - prominent, moderate |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 0 |
(n=5) 1 |
(n=5) 0 |
(n=5) 0 |
(n=1) 0 |
Mammary gland - thick, moderate |
(n=10) 0 |
(n=10) 0 |
(n=10) 0 |
(n=9) 0 |
(n=8) 0 |
(n=10) 1 |
(n=8) 0 |
(n=1) 0 |
* P<0.05, ** P<0.01
# excluded from dataset
Table 7 Absolute and adjusted organ weights (F0)
Doses (mg/kg/day) |
Control |
100 |
300 |
500 |
Control |
100 |
300 |
500 |
MALE |
FEMALE |
|||||||
BODY WEIGHT |
||||||||
Weight (g) |
391.8 |
407.9 |
381.0 |
356.0* |
249.8 |
253.8 |
252.2 |
|
TESTES |
||||||||
- Absolute weight (g) |
3.561 |
3.534 |
3.724 |
3.781 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.912 |
0.878 |
0.982 |
1.068 |
n/a |
n/a |
n/a |
n/a |
EPIDIDYMIS |
||||||||
- Absolute weight (g) |
1.413 |
1.320 |
1.431 |
1.346 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.362 |
0.327 |
0.377 |
0.381 |
n/a |
n/a |
n/a |
n/a |
SEMINAL VESICLE |
||||||||
- Absolute weight (g) |
0.843 |
0.816 |
0.827 |
0.805 |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.216 |
0.199 |
0.214 |
0.228 |
n/a |
n/a |
n/a |
n/a |
PROSTATE |
||||||||
- Absolute weight (g) |
1.002 |
0.957 |
0.969 |
0.776*** |
n/a |
n/a |
n/a |
n/a |
- % body weight |
0.256 |
0.236 |
0.254 |
0.219** |
n/a |
n/a |
n/a |
n/a |
BRAIN |
||||||||
- Absolute weight (g) |
2.071 |
2.069 |
2.015 |
2.026 |
1.883 |
1.946 |
1.961 |
nd |
- % body weight |
0.519 |
0.484 |
0.526 |
0.585* |
0.767 |
0.733 |
0.768 |
nd |
ADRENAL |
||||||||
- Absolute weight (g) |
0.070 |
0.069 |
0.070 |
0.076 |
0.086 |
0.076 |
0.093 |
nd |
- % body weight |
0.018 |
0.016 |
0.018 |
0.022 |
0.035 |
0.029* |
0.036 |
nd |
SPLEEN |
||||||||
- Absolute weight (g) |
0.605 |
0.651 |
0.554 |
0.526 |
0.577 |
0.504 |
0.491 |
nd |
- % body weight |
0.152 |
0.152 |
0.144 |
0.152 |
0.235 |
0.189** |
0.191** |
nd |
THYMUS |
||||||||
- Absolute weight (g) |
0.390 |
0.468 |
0.343 |
0.332 |
0.231 |
0.274 |
0.212 |
nd |
- % body weight |
0.098 |
0.109 |
0.090 |
0.096 |
0.093 |
0.104 |
0.082 |
nd |
LIVER |
||||||||
- Absolute weight (g) |
9.050 |
10.658** |
11.517** |
12.538** |
9.184 |
8.927 |
10.809 |
nd |
- % body weight |
2.272 |
2.489 |
2.993* |
3.594*** |
3.759 |
3.346 |
4.206 |
nd |
HEART |
||||||||
- Absolute weight (g) |
1.102 |
1.078 |
1.002 |
0.938*** |
0.859 |
0.798 |
0.849 |
nd |
- % body weight |
0.277 |
0.252 |
0.261 |
0.269 |
0.353 |
0.301 |
0.331 |
nd |
KIDNEY |
||||||||
- Absolute weight (g) |
2.257 |
2.473 |
2.717 |
2.426 |
1.841 |
1.747 |
1.864 |
nd |
- % body weight |
0.567 |
0.576 |
0.707* |
0.699* |
0.755 |
0.656 |
0.723 |
nd |
PITUITARY |
||||||||
- Absolute weight (g) |
0.010 |
0.009 |
0.010 |
0.009 |
0.012 |
0.012 |
0.013 |
nd |
- % body weight |
0.003 |
0.002 |
0.002 |
0.003 |
0.005 |
0.005 |
0.005 |
nd |
THYROID/ PARA |
||||||||
- Absolute weight (g) |
0.023 |
0.023 |
0.026 |
0.024 |
0.019 |
0.019 |
0.022 |
nd |
- % body weight |
0.006 |
0.006 |
0.007** |
0.007* |
0.008 |
0.007 |
0.009 |
nd |
OVARY |
||||||||
- Absolute weight (g) |
n/a |
n/a |
n/a |
n/a |
0.110 |
0.112 |
0.112 |
nd |
- % body weight |
n/a |
n/a |
n/a |
n/a |
0.044 |
0.044 |
0.044 |
nd |
UTERUS |
||||||||
- Absolute weight (g) |
n/a |
n/a |
n/a |
n/a |
0.501 |
0.457 |
0.496 |
nd |
- % body weight |
n/a |
n/a |
n/a |
n/a |
0.203 |
0.181 |
0.197 |
nd |
* P<0.05, ** P<0.01, ***p<0.001
nd: not determined
Table 8 Summary of mean estrous cycle data (n=10)
Predose |
Pre-pairing |
||||||||||||||
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
||||||||
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
No. of estrous cycles |
Cycle length (days) |
2.3 |
4.5 |
2.5 |
4.1 |
2.1 |
4.9 |
2.2 |
4.4 |
2.4 |
4.5 |
2.2 |
4.1 |
2.4 |
4.2 |
2.2 |
5.0 |
Table 9 Summary of male fertility and reproductive performance
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Total males |
10 |
10 |
10 |
10 |
- Unscheduled deaths prior to cohabitation |
0 |
0 |
0 |
0 |
Males cohabitated |
10 |
10 |
10 |
10 |
- Unscheduled deaths during cohabitation |
0 |
0 |
0 |
1 |
Males mating with at least 1 female |
10 |
10 |
10 |
9 |
Males impregnating at least 1 female |
9 |
10 |
9 |
9 |
Mating index (%) |
100 |
100 |
100 |
90 |
Fecundity index (%) |
90 |
100 |
90 |
100 |
Fertility index (%) |
90 |
100 |
90 |
90 |
Mating index % = (no. of males mating with at least 1 female / no. of males cohabitated with at least 1 female) x 100
Fecundity index % = (no. of males impregnating at least 1 female / no. of males mating with at least 1 female) x 100
Fertility index % = (no. of males impregnating at least 1 female / no. of males cohabited with at least 1 female) x 100
Table 10 Summary of female fertility and reproductive performance
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Total females |
10 |
10 |
10 |
10 |
- Unscheduled deaths prior to cohabitation |
0 |
0 |
0 |
0 |
Females cohabitated |
10 |
10 |
10 |
10 |
- Unscheduled deaths during cohabitation |
0 |
0 |
0 |
0 |
Females mated |
10 |
10 |
10 |
10 |
Pregnant females |
9 |
10 |
9 |
10 |
- Pup(s) / total litter dead |
1 |
0 |
1 |
8 |
Non-pregnant females |
1 |
0 |
1 |
0 |
Mating per day periods of cohabitation: |
||||
- Day 1 |
2 |
4 |
1 |
1 |
- Day 2 |
2 |
0 |
2 |
1 |
- Day 3 |
4 |
2 |
3 |
3 |
- Day 4 |
2 |
4 |
2 |
3 |
- Day 7 |
0 |
0 |
1 |
2 |
- Day 12 |
0 |
0 |
1 |
0 |
Mating index % |
100 |
100 |
100 |
100 |
Fecundity index % |
90 |
100 |
90 |
100 |
Fertility index % |
90 |
100 |
90 |
100 |
Mating index % = (mated females / females cohabitated (exc. females sacrificed during cohabitation)) x 100
Fecundity index % = (pregnant females / mated females (exc. females with an undetermined pregnancy status)) x 100
Fertility index % = (pregnant females / females cohabitated (exc. females sacrificed during cohabitation or with an undetermined pregnancy status)) x 100
Effect on fertility: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 100 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The endpoint is concluded based on a single key study with a Klimisch rating of 1. The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.
A summary of adult reproductive responses to the test item are described below;
Males
Reproductive performance was not affected by the test article i.e. no test-article related effects on mating or fertility were noted.
Females and litter
Nine of 10 females administered 500 mg/kg/day produced a litter, of which one entire litter was stillborn. For females administered 500 mg/kg/day, between 22 to 100 % of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. Eight litters were recorded with live pups at birth, with total litter losses noted by PND 2 for four of these litters, with only one litter surviving to scheduled necropsy on PND 13.
The litter death was attributed to the test article and was considered adverse.
A test article-related effect on the live birth index was observed following 300 mg/kg/day administration (83.4% compared to 100% for controls), however, post-implantation survival index and pup survival indices were comparable with controls and sex ratio was unaffected
Endpoint discussion
Test article-related findings observed in males following 500 mg/kg/day administration were generally confined to systemic toxicity in the rat. These findings were considered not to represent an adverse effect in reproduction, and hence, the no observed effect level (NOEL) for reproductive toxicity in males was established as 500 mg/kg/day. The NOEL for females reproductive toxicity was established as 100 mg/kg/day based on the number of litter losses observed in the 500 mg/kg/day test group and the test article-related effect on the live birth index following 300 mg/kg/day administration.
Effects on developmental toxicity
Description of key information
NOEL and NOAEL (developmental toxicity) = 100 mg/kg bw/d; OECD 422; Barraclough, 2018
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 July 2017 - 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- other: CRL:WI(Han)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:
Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported, assume yes
- Age at study initiation:
10-11 weeks old
- Weight at study initiation:
Males: 288.6 - 391.8 g; females: 173.2 - 235.3 g
- Fasting period before study:
No
- Housing:
During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum):
Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum):
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
- Acclimation period:
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 days prior to initiation of smearing (females).
ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 ºC
- Humidity (%):
30-70 % RH
- Air changes (per hr):
15-20
- Photoperiod (hrs dark / hrs light):
12:12
IN-LIFE DATES: From: 26 July 2017 To: 25 September 2017 - Route of administration:
- oral: gavage
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
##
- VEHICLE
- Justification for use and choice of vehicle (if other than water):
Homogenous stable solutions were prepared in corn oil, a guideline recommended vehicle.
- Concentration in vehicle:
##
- Amount of vehicle (if gavage):
5 mL/kg
- Lot/batch no. (if required):
MKBP7039V, MKBZ9899V and MKCC0462
- Purity: Assumed pure corn oil - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Single 5.0 mL aliquot formulation samples were collected from the main in-life experiment study for each animal group at the respective concentrations (0, 20.0, 60.0 and 100.0 mg/mL) on the 26th July 2017 (week 1 of the in-life phase), 9th August 2017 (week 3 of the in-life phase) and 30th August 2017 (week 6 of the in-life phase) for analysis.
A validated gradient elution reversed-phase high-performance liquid chromatographic method with ultraviolet detection (HPLC-UV) was employed for determination of formulation stability and homogeneity, and concentration analyses of formulations administered during the study.
No test item was observed in the control group samples. Measured concentrations from the test item groups were found to be in the range of 89.2 - 102.3 % of nominal values. - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: Up to 15 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 (GD 0) of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility for up to an additional 5 days.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Females were housed individually during gestation, and with their litter during the lactation phase.
- Any other deviations from standard protocol: No - Duration of treatment / exposure:
- Males: 42 consecutive days (2 weeks prior to pairing; during pairing; and until the day before necropsy)
Females: 61 days (2 weeks prior to pairing [pre-pairing phase]; during the pairing phase; and until Lactation Day (LD) 13, inclusive, or 25 days post-coitum for females which did not litter and were sent to necropsy on LD 14 or 26 days post coitum). Some females were not dosed on LD 0 if they were observed to be starting or just completed parturition. - Frequency of treatment:
- Daily
- Duration of test:
- Males: 42 consecutive days (2 weeks prior to pairing; during pairing; and until the day before necropsy)
Females: 61 days (2 weeks prior to pairing [pre-pairing phase]; during the pairing phase; and until Lactation Day (LD) 13, inclusive, or 25 days post-coitum for females which did not litter and were sent to necropsy on LD 14 or 26 days post coitum). Some females were not dosed on LD 0 if they were observed to be starting or just completed parturition. - Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10 males and 10 females per treatment group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses selected based on the results of a preliminary range-finder test.
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
BODY WEIGHT: Yes
- Time schedule: Male body weights were recorded once during acclimation, before dosing on the first day of dosing, at weekly intervals, and before necropsy. Female body weights were recorded once during acclimation; before dosing on the first day of dosing; at weekly intervals prior to pairing and until confirmation of mating; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 1, 4, 13 and 14 (prior to necropsy).
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 0 to 13. Consumption was calculated as g/animal/day.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The amount of water consumed was determined daily during the pre-pairing phase (both sexes). Consumption was calculated as g/animal/day.
OTHER: N/A - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: N/A - Fetal examinations:
- N/A
- Statistics:
- Data for each sex were analyzed separately, unless stated otherwise. Only data collected on or after the first day of dosing were analyzed statistically. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P ≤ 0.05. By default, significant results were reported as *P ≤ 0.05, +P ≤ 0.01, and/or #P ≤ 0.001.
- Indices:
- The duration of gestation
Number of implantation sites
Number of pups born
Number of pups alive on Post Natal Day (PND) 1, 4 (before culling)
% male pups on PND 1
Percent post-implantation loss
Live birth and survival indices PND 1-4
Pup weights
Ano-genital distance (male only)
Thyroid hormones - Historical control data:
- Reffered to but details not reported
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- An observation of vocalizing on handling was noted in one female from each test article treated group from Pre-Pairing Day 4 or 5. One female administered 500 mg/kg/day was noted to have wet fur of the anogenital area and/or abdomen during the pre-pairing phase or GD 7. These observations were attributed to test article.
Incidences of mouth rubbing were noted immediately upon the return to the home cage in up to 4 of 10 females administered 500 mg/kg/day. Salivation was observed from Pre-Pairing Day 8 for some females administered 100, 300, or 500 mg/kg/day and persisted through pairing for occasional females, but no dose response was noted. Salivation was not recorded during gestation or lactation. These observations were attributed to the consistency and palatability of the test article and were considered non-adverse. - Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- N/A
- Mortality:
- no mortality observed
- Description (incidence):
- No maternal female mortality was observed.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weight gain was for females administered 500 mg/kg/day, when compared with controls during late gestation.
Mean body weight during the pre-pairing, gestation, or lactation phases for females administered 100, 300 mg/kg/day were compared with controls and considered unaffected by the test article. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Lower food consumption was noted for males and females administrated 500 mg/kg/day during the first two weeks of dosing. Food consumption was also noted to be lower for 500 mg/kg/day females during late gestation, when compared with controls.
No effect on food consumption was noted following 100 or 300 mg/kg/day administration. - Food efficiency:
- not examined
- Description (incidence and severity):
- N/A
- Description (incidence and severity):
- A test article related effect on water consumption was noted for animals administered 300 or 500 mg/kg/day.
Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, compared with controls, such that at the end of the pre-pairing phase, female water consumption was 38 or 80% greater than controls, respectively. - Ophthalmological findings:
- not examined
- Description (incidence and severity):
- N/A
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Haematological parameters were unaffected by the test item.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Cholesterol and glucose levels were statisticaly significantly elevated in females administered 300 mg/kg/day. Data from the 500 mg/kg/day female was also elevated in comparison to the control however the data was excluded from statistical analysis as only 1 female was analysed.
All other values, including those which attained statistical significance, were considered incidental and not related to Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) as no dose relationship was noted and/or values were within background ranges. - Urinalysis findings:
- not examined
- Description (incidence and severity):
- N/A
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- Female changes during the assessments included piloerection and salivation, which were generally dose related and behavior changes predominantly characterized by paw flicking (fore paws). A statistically significant decreased number of rears were noted on Lactation Day (LD) 1 and 7 for females 300 mg/kg/day, compared with controls. A lower number was also recorded on LD 1 for females administered 500 mg/kg/day; these females were early decedents due to litter loss and, therefore, no data are available for LD 7.
- Immunological findings:
- not examined
- Description (incidence and severity):
- N/A
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Organ weight parameters were unaffected by the test item.
- Gross pathological findings:
- not specified
- Description (incidence and severity):
- Small thymus was recorded in one female administered 300 mg/kg/day.
- Neuropathological findings:
- no effects observed
- Description (incidence and severity):
- Locomotor activity was unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In the liver, centrilobular hepatocyte hypertrophy was recorded in all examined females administered 300 or 500 mg/kg/day. This finding was characterized by the enlargement of the hepatocytes, a ground glass appearance of the cytoplasm, and slight compression of the adjacent sinusoids; was principally localized within the centrilobular areas, but occasionally also extended more diffusely to midzonal and periportal regions in affected animals; and correlated generally with increased group mean adjusted liver weights and the macroscopic finding of dark/mottled discoloration.
In the thyroid, follicular cell hypertrophy was recorded in four females administered 300 mg/kg/day and one female administered 500 mg/kg/day and was characterized by follicular cells with increased amounts of cytoplasm, follicular epithelium that ranged from flat to cuboidal or columnar and follicles with decreased amounts of colloid. - Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- No tumours were observed.
- Other effects:
- not examined
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- No incident of abortion was observed.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- Implantation loss (%) was unaffected by the test item.
- Total litter losses by resorption:
- not specified
- Description (incidence and severity):
- One female treated at 500 mg/kg/day did not litter and was found to have 10 implantation sites with no evidence of foetus in the uterus. This is indicative of 100 % early resorption.
The report did not discuss if this was a test item related response. - Early or late resorptions:
- not specified
- Description (incidence and severity):
- One female treated at 500 mg/kg/day did not litter and was found to have 10 implantation sites with no evidence of foetus in the uterus. This is indicative of 100 % early resorption.
The report did not discuss if this was a test item related response. - Dead fetuses:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article-related effects on litter survival were noted. Eight of 10 females administered 500 mg/kg/day were sent to necropsy between LD 0 and 2 due to total litter loss. In all females that successfully littered, up to 100% of pups were still born, with three litters which contained still born pups only. The litter death was attributed to the test article and was adverse.
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Pregnancy duration was not effected by the test item.
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- One control female and one female administered 300 mg/kg/day were sent to necropsy on Gestation Day (GD) 26 having failed to produce a litter. At necropsy, the animals were found to be non-pregnant. The non-pregnant finding for these animals was considered incidental, and no relationship to dose was noted.
- Details on maternal toxic effects:
- No test article-related effects on clinical or postdose observations were noted, although lower body weight gain and food consumption was noted during late gestation following 500 mg/kg/day administration.
Some markers of toxicity were observed. The FOB observations included piloerection, salivation, and paw flicking (fore paws), which increased in a dose dependent manner. A decreased number of rears was also noted during the lactation phase for females 300 mg/kg/day, compared with controls. Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, at 38 or 80% greater than the controls, respectively. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day. However, the macroscopic and/or microscopic observations noted in the kidney of males were not apparent in females.
Microscopic observations included epidermal hyperplasia and/or hyperkeratosis in the nonglandular stomach, which were considered consistent with a chronic irritation-type reaction.
Test article-related effects on litter survival were noted. Eight of 10 females administered 500 mg/kg/day were sent to necropsy between LD 0 and 2 due to total litter loss. In all females that successfully littered, up to 100% of pups were still born, with three litters which contained still born pups only. The litter death was attributed to the test article and was adverse.
Hepatocyte hypertrophy in the liver and the associated increase in TSH and thyroid follicular cell hypertrophy were less pronounced in females than males; nevertheless, it is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites and is considered rat specific. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- behaviour (functional findings)
- body weight and weight gain
- clinical biochemistry
- dead fetuses
- food consumption and compound intake
- water consumption and compound intake
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A test article-related effect on body weight gain was noted for pups from litters of dams administered 300 or 500 mg/kg/day, compared with controls.
Only two litters survived to PND 1 and only one litter survived to PND 13 due to the number of total litter losses between PND 0 and 2 observed for litters of dams administered 500 mg/kg/day. Of the few surviving litters, a reduction in body weight was noted, compared with controls. Where statistical evaluation was possible (PND 1), this reduction attained a statistical significance. - Reduction in number of live offspring:
- effects observed, treatment-related
- Description (incidence and severity):
- Nine of 10 females administered 500 mg/kg/day produced a litter. Five litters were recorded to have live pups at birth, with a single litter surviving to scheduled necropsy; in this litter only 4 of 12 pups alive on PND 1. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. One litter contained still born pups only, and, excluding one litter which survived to PND 13, all others had total litter death by PND 2.
An effect was observed for the live birth index of females administered 300 mg/kg/day compared with controls (83.4% compared to 100% for control), but postnatal survival was comparable with control values. - Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- effects observed, treatment-related
- Description (incidence and severity):
- A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in males (PND1, 4, 7 and 13) and females (PND 1, 4 and 7).
No test article-related effect was noted in litters of dams administered 100 mg/kg/day. - Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- One litter from a control dam and one litter from a dam administered 300 mg/kg/day died by PND 2. The 300 mg/kg/day animal had 12 pups in total, two still born pups and all remaining pups found dead with no milk evident in the stomach on PND 0. The control animal had 1 pup in total, which was noted to be cold to touch, weak, unfed, and was found dead on PND 2. The deaths were considered incidental as they were noted in controls or occurred as a single incident in the group, with no other effects on pup survival.
- External malformations:
- no effects observed
- Description (incidence and severity):
- Only one 500 mg/kg/day litter survived to PND 4 due the high incidence of litter death; as such, this may have been incidental and not related to test article even though values were lower. No effect on ano-genital distance was observed in litters of dams administered 100 or 300 mg/kg/day.
No nipple/areolae were observed for males surviving to PND 13. - Skeletal malformations:
- not examined
- Visceral malformations:
- not examined
- Other effects:
- not examined
- Details on embryotoxic / teratogenic effects:
- Excluding the total litter death noted for the litters of dams administered 500 mg/kg/day, no other test article-related pup clinical observations were noted.
A test article-related effect on body weight and body weight gain was noted for pups from litters of dams administered 300 or 500 mg/kg/day, compared with controls.
Only two litters survived to PND 1 and only one litter survived to PND 13 due to the number of total litter losses between PND 0 and 2 observed for litters of dams administered 500 mg/kg/day. Of the few surviving litters, a reduction in body weight was noted, compared with controls. Where statistical evaluation was possible (PND 1), this reduction attained a statistical significance.
A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in males, females, and/or combined values on all body weight occasions. A reduction in combined body weight gain of 2.9 g was also noted between PND 1 to 13.
No external malformations or effects on thyroid hormone levels was noted at any treatment level.
In summary, no test article-related effect was noted in litters of dams administered 100 mg/kg/day. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- reduction in number of live offspring
- fetal/pup body weight changes
- changes in litter size and weights
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 300 mg/kg bw/day (nominal)
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects as a secondary non-specific consequence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Conclusions:
- No test article-related changes were noted in the litters following parental administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for developmental toxicity was established as 100 mg/kg/day.
- Executive summary:
OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.
A summary of developmental toxicity the test item are described below;
Litters and offspring
Nine of 10 females administered 500 mg/kg/day produced a litter, of which one entire litter was stillborn. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. Eight litters were recorded with live pups at birth, with total litter losses noted by PND 2 for four of these litters, with only one litter surviving to scheduled necropsy on PND 13. The litter death was attributed to the test article and was considered adverse.
A test article-related effect on the live birth index was observed following 300 mg/kg/day administration (83.4% compared to 100% for controls), however, post-implantation survival index and pup survival indices were comparable with controls and sex ratio was unaffected
A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in male pups (PND 1, 4, 7 and 13) and female pups (PND 1, 4 and 7). No test article-related effect was noted in litters of dams administered 100 mg/kg/day.
Ano-genital distance, pup thyroid hormone levels, and male nipple/areolae count were unaffected by the test article.
Endpoint discussion
No test article-related changes were noted in the litters following parental administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for developmental toxicity was established as 100 mg/kg/day.
Reference
Table 5 Summary of Parturition and Litter Data
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
|
Duration of gestation (days) |
Mean |
23.0 |
23.1 |
22.6 |
23.1 |
N |
9 |
10 |
9 |
9 |
|
No. of implantation sites |
Mean |
11.3 |
10.6 |
12.3 |
12.3 |
N |
9 |
10 |
9 |
9 |
|
No. of pups born |
Mean |
10.7 |
9.2 |
11.7 |
10.0 |
N |
9 |
10 |
9 |
9 |
|
No. of pups alive at Post-Natal Day 1 |
Mean |
0.7 |
9.2 |
9.7 |
0.8*** |
N |
9 |
10 |
9 |
9 |
|
% male pups at PND 1 |
Mean |
49.4 |
47.4 |
59.0 |
29.2 |
N |
9 |
10 |
8 |
2 |
|
No. of pups alive at PND 4 before culling |
Mean |
10.6 |
9.1 |
10.9 |
2.0 |
N |
9 |
10 |
8 |
1 |
|
No. of pups culled at PND 4 |
Mean |
1.9 |
1.0 |
1.6 |
0.0 |
N |
8 |
10 |
8 |
1 |
|
No. of pups alive after PND 4 culling |
Mean |
10.0 |
8.1 |
9.3 |
4.0 |
N |
8 |
10 |
8 |
1 |
|
No. of pups alive at PND 7 |
Mean |
10.0 |
8.0 |
9.3 |
4.0 |
N |
8 |
10 |
8 |
1 |
|
No. of pups alive at PND 13 |
Mean |
10.0 |
8.0 |
9.3 |
4.0 |
N |
8 |
10 |
8 |
1 |
|
*** p<0.001
Table 6 Summary of pup survival
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
|
Post-implantation survival index % |
Mean |
94.7 |
87.7 |
94.6 |
81.4 |
N |
9 |
10 |
9 |
9 |
|
Live birth index % |
Mean |
100.0 |
100.0 |
83.4 |
7.4*** |
N |
9 |
10 |
9 |
9 |
|
Survival index PND 1-4 % |
Mean |
88.9 |
95.0 |
100.0 |
50.0 |
N |
9 |
10 |
8 |
2 |
|
Survival index PND 4-7 % |
Mean |
100.0 |
99.0 |
100.0 |
100.0 |
N |
8 |
10 |
8 |
1 |
|
Survival index PND 7-13 % |
Mean |
100.0 |
100.0 |
100.0 |
100.0 |
N |
8 |
10 |
8 |
1 |
|
*** p <0.001
Table 7 Summary of pup clinical observations
Clinical observation |
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
Bruised tip of tail |
- |
- |
1 |
- |
Cold |
1 |
- |
1 |
1 |
Found dead |
1 |
1 |
2 |
8 |
Hemorrhagic area to face |
- |
1 |
- |
- |
Missing, presumed cannibalized |
- |
- |
1 |
5 |
Thinning fur |
- |
1 |
- |
- |
Total litter loss |
1 |
- |
1 |
8 |
Umbilical cord attached |
- |
- |
1 |
- |
Unfed |
1 |
1 |
- |
- |
Weak |
1 |
1 |
- |
- |
Table 8 Summary of pup body weights
|
Control |
100 mg/kg/day |
300 mg/kg/day |
500 mg/kg/day |
||
Post-Natal Day 1 |
Male |
Adjusted mean |
7.2 |
7.1 |
5.9** |
5.1*** |
Mean |
7.2 |
7.1 |
5.9 |
5.1 |
||
N |
8 |
10 |
8 |
2 |
||
Female |
Adjusted mean |
6.6 |
6.9 |
5.6** |
5.3* |
|
Mean |
6.6 |
6.8 |
5.7 |
5.3 |
||
N |
9 |
10 |
8 |
2 |
||
Combined |
Adjusted mean |
6.8 |
7.0 |
5.7** |
5.3* |
|
Mean |
6.8 |
7.0 |
5.8 |
5.3 |
||
N |
9 |
10 |
8 |
2 |
||
Post-Natal Day 4 |
Male |
Adjusted mean |
11.0 |
11.0 |
8.8*** |
- |
Mean |
10.9 |
11.2 |
8.7 |
7.6 |
||
N |
8 |
9 |
8 |
1 |
||
Female |
Adjusted mean |
10.3 |
10.5 |
8.4* |
- |
|
Mean |
10.3 |
10.6 |
8.4 |
9.0 |
||
N |
8 |
10 |
8 |
1 |
||
Combined |
Adjusted mean |
10.6 |
10.7 |
8.6** |
- |
|
Mean |
10.6 |
10.7 |
8.6 |
8.7 |
||
N |
8 |
10 |
8 |
1 |
||
Post-Natal Day 7 |
Male |
Adjusted mean |
16.4 |
16.9 |
13.2** |
- |
Mean |
16.4 |
17.0 |
13.2 |
5.4 |
||
N |
8 |
9 |
8 |
1 |
||
Female |
Adjusted mean |
15.5 |
16.2 |
12.9* |
- |
|
Mean |
15.6 |
16.0 |
13.0 |
7.1 |
||
N |
8 |
10 |
8 |
1 |
||
Combined |
Adjusted mean |
15.9 |
16.4 |
13.0** |
- |
|
Mean |
16.0 |
16.2 |
13.1 |
6.7 |
||
N |
8 |
10 |
8 |
1 |
||
Post-Natal Day 13 |
Male |
Adjusted mean |
28.4 |
29.7 |
24.0** |
- |
Mean |
28.4 |
29.7 |
24.0 |
27.2 |
||
N |
8 |
9 |
8 |
1 |
||
Female |
Adjusted mean |
27.1 |
28.5 |
23.4 |
- |
|
Mean |
27.5 |
28.0 |
23.7 |
29.1 |
||
N |
8 |
10 |
8 |
1 |
||
Combined |
Adjusted mean |
27.5 |
28.8 |
23.5 |
- |
|
Mean |
27.9 |
28.3 |
23.8 |
29.1 |
||
N |
8 |
10 |
8 |
1 |
||
* p<0.05
** p<0.01
***p <0.001
Table 9 Summary of ano-genital distance (mm) at PND 4
|
Male |
Female |
|
Control |
Adjusted mean |
3.6 |
- |
Mean |
3.8 |
2.0 |
|
N |
8 |
8 |
|
100 mg/kg/day |
Adjusted mean |
3.9 |
- |
Mean |
4.1 |
2.0 |
|
N |
9 |
10 |
|
300 mg/kg/day |
Adjusted mean |
4.0 |
- |
Mean |
3.5 |
1.8 |
|
N |
7 |
7 |
|
500 mg/kg/day |
Adjusted mean |
- |
- |
Mean |
3.0 |
1.0 |
|
N |
1 |
1 |
|
Table 10 Summary of male nipples/ areolae at PND 13
|
No. of litter examined |
Count |
Control |
8 |
0 |
100 mg/kg/day |
9 |
0 |
300 mg/kg/day |
8 |
0 |
Table 11 Summary of pup thyroid analysis at PND 13
|
Male |
Female |
Combined |
|||||||
IMT4 (nmol/L) |
TSHI (µIU/mL) |
TT3S (nmol/L) |
IMT4 (nmol/L) |
TSHI (µIU/mL) |
TT3S (nmol/L) |
IMT4 (nmol/L) |
TSHI (µIU/mL) |
TT3S (nmol/L) |
||
Control |
Adjusted mean |
83 |
0.31 |
0.89 |
88 |
0.27 |
0.99 |
86 |
0.29 |
0.94 |
Mean |
11.6 |
0.122 |
0.174 |
12.8 |
0.086 |
0.110 |
11.6 |
0.088 |
0.123 |
|
N |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
|
100 mg/kg/day |
Adjusted mean |
96 |
0.23 |
1.13* |
95 |
0.26 |
1.08 |
95 |
0.24 |
1.12* |
Mean |
10.7 |
0.106 |
0.171 |
15.2 |
0.165 |
0.175 |
11.7 |
0.128 |
0.167 |
|
N |
9 |
9 |
9 |
10 |
10 |
10 |
10 |
10 |
10 |
|
300 mg/kg/day |
Adjusted mean |
90 |
0.19 |
1.00 |
91 |
0.22 |
0.92 |
91 |
0.21 |
0.96 |
Mean |
13.5 |
0.043 |
0.143 |
20.0 |
0.051 |
0.121 |
14.8 |
0.033 |
0.119 |
|
N |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
8 |
|
500 mg/kg/day |
Adjusted mean |
87E |
0.17E |
1.18E |
96E |
0.28E |
1.36E |
92E |
0.23E |
1.27E |
Mean |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
N |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
|
IMT4: Imulite Total T4
TSHI: Rapid Thyroid Stimulating Hormone
TT3S: Total T3
E: excluded from dataset
* p<0.05
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 100 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The endpoint is concluded based on a single key study with a Klimisch rating of 1. The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band.
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
OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.
A summary of developmental toxicity the test item are described below;
Females and offspring
Nine of 10 females administered 500 mg/kg/day produced a litter, of which one entire litter was stillborn. For females administered 500 mg/kg/day, between 22 to 100% of pups in the litter were stillborn; pups found dead presented clinically with no evidence of milk in the stomach or were cannibalized. Eight litters were recorded with live pups at birth, with total litter losses noted by PND 2 for four of these litters, with only one litter surviving to scheduled necropsy on PND 13. The litter death was attributed to the test article and was considered adverse.
A test article-related effect on the live birth index was observed following 300 mg/kg/day administration (83.4% compared to 100% for controls), however, post-implantation survival index and pup survival indices were comparable with controls and sex ratio was unaffected
A reduction in mean body weight values from litters of dams administered 300 mg/kg/day was noted from PND 1 to 13 (between 80 to 86%). This attained statistical significance in male pups (PND 1, 4, 7 and 13) and female pups (PND 1, 4 and 7). No test article-related effect was noted in litters of dams administered 100 mg/kg/day.
Ano-genital distance, pup thyroid hormone levels, and male nipple/areolae count were unaffected by the test article.
Endpoint discussion
No test article-related changes were noted in the litters following parental administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for developmental toxicity was established as 100 mg/kg/day.
Mode of Action Analysis / Human Relevance Framework
During the treatment period, the main adverse effects observed at mid and high dose groups were local effects of the test item resulting into epithelial hyperplasia and orthokeratotic hyperkeratosis recorded in the stomach, which correlate with the increased mean water consumption. Similarly effects were noted in offspring at these dose levels, consisting of.dose-dependent incidence of stillborn pups and lower mean body weights in surviving pups. These effects are considered relevant for humans.
Justification for classification or non-classification
The substance does not meet the criteria for classfication under reproductive toxicity in accordance with GHS and Regulation (EC) No 1272/2008 (CLP).
Additional information
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