Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Reliability changed from "1" to "2" according to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010).
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: aqueous solution
Details on test material:
- Name of test material (as cited in study report): Generic name: Alkyl (C12-C16) dimethyl ammonio acetate
- Physical state: colourless to pale yellow liquid
- Analytical purity: see 'Composition of test material, percentage of components'
- Composition of test material, percentage of components: CAS No. 683-10-3: Dodecanaminium, N-(carboxymethyl)-N,N-dimethyl-, inner salt (19.84%); CAS No. 2601-33-4: Tetradecanaminium, N-(carboxymethyl)-N,N, inner salt (6.72%)
- Purity test date: 22 January 2008
- Lot/batch No.: W17C078716
- Expiration date of the lot/batch: 01 March 2008
- Stability under test conditions: Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer, dose formulations were prepared weekly. The application formulations were considered to be stable for at least 7 days when kept at room temperature
- Storage condition of test material: room temperature (20±5°C), away from direct sunlight
- Other: pH 7.02 (1% solution)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Fuellinsdorf, Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: males: 296 - 330 g; females: 180 - 212 g
- Fasting period before study: no data
- Housing: individually; during the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles. During the pairing period, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if: a) The daily vaginal smear was sperm positive, or b) A copulation plug was observed.
- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 (batch no. 65/07 rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), ad libitum.
- Water (e.g. ad libitum): Community tap-water from Fuellinsdorf, ad libitum in water bottles.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose levels were in terms of the a.i. in the aqueous solution. A correction factor of 3.765 was used.

The dose formulations were prepared weekly, using the test item as supplied by the Sponsor. The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.


VEHICLE
- Concentration in vehicle: 5, 15, 30 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of copulation
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution of the test substance in milli-Q-water was demonstrated. The application formulations were considered to be stable for at least 7 days when kept at room temperature.
Duration of treatment / exposure:
Males: Minimum 4 weeks; females: Approximately 7 weeks
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 300 mg/kg bw/day (acitve ingredient)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a previous non-GLP dose range finding toxicity study in Han Wistar Rats, RCC Study Number B62763.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, during acclimatization and up to day of necropsy; viability/mortality twice daily
- Cage side observations included: clinical signs; females additionally for signs of difficult or prolonged parturition and behavioural abnormalities in nesting and nursing


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and daily thereafter. Observations included: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.


BODY WEIGHT: Yes
- Time schedule for examinations: Daily from treatment start to day of necropsy.


FOOD CONSUMPTION:
Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-pairing period days 1-8 and 8-14; gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum. No food consumption was recorded during the pairing period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 males from each group, 5 lactating females from each group
- Parameters examined: Complete Blood Cell Count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count (total), Differential leukocyte count, Platelet count, Coagulation, Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day before or on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Animals fasted: Yes, approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: 5 males from each group, 5 lactating females from each group
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin (total), Cholesterol (total), Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase, Bile acids, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein (total), Albumin, Globulin, Albumin/Globulin ratio


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 P generation males shortly before the scheduled sacrifice and 5 P generation females on day 3 or 4 post partum, following the daily dose administration
- Dose groups that were examined: each group
- Battery of functions tested:
a) Cage-side observations: unusual body movements (e.g. tremors, convulsions), abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex and reaction to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (were made at any time during the FOB): hair coat, behavior, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

OTHER:
Mating performance and fertility, duration of gestation
Oestrous cyclicity (parental animals):
During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrous cycles.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
The testes and epididymides of all parental males were weighed as pairs. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Litter observations:
STANDARDISATION OF LITTERS
Not performed, pups were sacrificed on day 4.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litter size, live births, still births, postnatal loss and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.


GROSS EXAMINATION OF DEAD PUPS:
yes, dead pups were examined macroscopically; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects.
- Maternal animals: Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77 ®). All P generation animals were exsanguinated.


GROSS NECROPSY
All parent animals were examined macroscopically for any structural changes. Special attention was directed at the organs of the reproductive system. The number of implantation sites, implantation rate, post-implantation loss, and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS
The testes and epididymides of all parental males were weighed as pairs. In addition, from 5 males and females selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: Adrenal glands (weighed as pairs), Brain, Heart, Kidneys (weighed as pairs), Liver, Thymus, Spleen.

The following tissues from all parental males and females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative), Ovaries.

In addition, from the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions, Brain, Spinal chord, Small and large intestines (incl. Peyer’s patches), Stomach, Liver, Kidneys, Adrenals, Spleen, Heart, Thymus, Thyroids, Trachea and lungs (preserved by inflation with fixative and then immersion), Uterus (with vagina), Urinary bladder, Lymph nodes (mesenterial, mandibular), Peripheral nerve (sciatic), Bone marrow.

All organ and tissue samples to be examined were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis were stained by PAS-hematoxylin.
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high dose groups were examined. The same applied to all occurring gross lesions. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Due to possible test item-related findings noted in the high dose group, the kidneys, urinary bladder, stomach (forestomach and glandular stomach), cecum, colon, rectum, trachea, lungs, bone marrow and adrenal glands were examined also in the animals of the low and mid dose groups. Histological examination of ovaries was carried out on females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age. All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77 ®).
- These pups were examined macroscopically for any structural changes.

GROSS NECROPSY
- Gross necropsy consisted of macroscopic examinations, dead pups were examined macroscopically, as well.
Statistics:
Fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices were calculated from online recorded data. For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

The following statistical methods were used for food consumption, body weight, macroscopical findings, organ weights and reproduction data:
Means and standard deviations, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex. The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution. Fisher's exact-test was applied to the macroscopical findings.
Reproductive indices:
Fertility index/conception rate (percentage of females pregnant), gestation index (percentage of dams not delivering dead pups), mean duration of gestation, implantation rate (number of implantation sites compared to control), Post-implantation loss.
Offspring viability indices:
Litter size at first check, postnatal loss, sex ratio.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived until the scheduled necropsy.

300 mg/kg bw/d:
Incidences of ruffled fur (from second week onwards, slightly ruffled in 4 males and all females, and one male had ruffled fur over the whole body), diarrhea (3 males and 3 females in the second week), sedation (all animals in the second and third week), salivation and pushing their head through the bedding occurred from day 5/6 of the pre-pairing period up to the end of the treatment, either periodically or continuously in the males and females, as well as salivation in some individuals. Four males were in a bad condition for up to 5 days during the pre-pairing period. The feces from all males and 4 females were noted to be round and soft in comparison to the control group. One female had loose feces. Several puddles of urine were noted for all males and females. In addition, mean body temperature was statistically significantly reduced in both males and females. All these findings were considered to be test item-related.

150 mg/kg bw/d:
All males and females pushed their head through the bedding after application from day 7/8 of the pre-pairing period onwards. Male no. 27 salivated periodically during the prepairing and pairing periods. All males salivated during the last 3 or 4 days of the after pairing period. Female no. 65 had salivation during the last part of the gestation period. All of the above were considered to be test item-related, with the exception of pushing the head through the bedding. This was considered to be a sign of discomfort following application rather than a toxic effect of the test item.

Control group and 50 mg/kg bw/d:
No clinical signs or symptoms were noted for any animal in any group during the study.

BODY WEIGHT (PARENTAL ANIMALS)
Males (Pre-pairing, Pairing and After Pairing Periods):
300 mg/kg bw/d:
Body weight gain was statistically significantly reduced from day 2 until the end of the pre-pairing period and absolute body weight from day 3 onwards. Over the whole of the pre-pairing period, males increased in weight by 1.6% compared to 12.9% in the control group. During the pairing and after pairing periods, body weight gain was not considered to have been affected by treatment with the test item but absolute body weight remained statistically significantly reduced until the end of the study period.

150 mg/kg bw/d:
Mean body weight gain was slightly reduced over the pre-pairing period (+9.8% compared to 12.9%). Thereafter, it was similar to that of the control group. Absolute body weight became reduced in the pre-pairing period and remained so for the rest of the study compared to the control group but was at no time statistically significantly reduced.

50 mg/kg bw/d:
Mean body weight and body weight gain were similar to that of the control group.

Females (Pre-pairing, Pairing, Gestation and Lactation Periods):
300 mg/kg bw/d:
Mean body weight gain was statistically significantly reduced from day 2 until day 13 and absolute body weight from day 3 until day 11 of the pre-pairing period (+6.6% compared to +9.7% in the control group over days 1 - 14). Thereafter, it was similar to that of the control group, with the exception of days 14 - 21 of the gestation period (+13.1% compared to +29.5% in the control group). Mean absolute body weight was statistically significantly reduced from day 18 - 21 of the gestation period.

150 and 50 mg/kg bw/d:
Mean body weight and body weight gain were not considered to have been affected by treatment with the test item.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Males (Pre-pairing and After Pairing Periods):
300 mg/kg bw/d:
Mean food consumption was statistically significantly reduced during the first week of the pre-pairing period (-44.5% compared to the control group over days 1 - 8). This was considered to be related to treatment with the test item. Food consumption recovered thereafter.

150 mg/kg bw/d:
Mean food consumption was reduced over days 1 - 8 (-11.7% compared to the control group). Although this reduction was not statistically significant, it was considered to be a test item-related effect. It remained reduced thereafter but there was no dose-dependent reduction.

50 mg/kg bw/d:
Food consumption was similar to that of the control group during the whole of the study.

Females (Pre-pairing, Gestation and Lactation Periods):
300 mg/kg bw/:
Mean food consumption was statistically significantly reduced over days 1 - 8 of the pre-pairing period (-37.5% compared to the control group) but recovered in the second week. In the gestation period, food consumption was again statistically significantly reduced over days 14 - 21 (-16.4% compared to the control group) and over the whole of the lactation period (-23.8% compared to the control group). This was considered to be related to treatment with the test item.

150 mg/kg bw/d:
Mean food consumption was reduced, but not statistically significantly over days 1 - 8 of the pre-pairing period (-10.7% compared to the control group). Thereafter it recovered and was similar to that of the control group.

50 mg/kg bw/d:
Mean food consumption was similar to the control group for the whole of the study.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All females mated within the first mating period. No test item-related effects were noted on the mean precoital time. Two females in the control group were not pregnant (fertility index and conception rate of 80%), as well as 1 female in the low dose group (fertility index and conception rate were 90%). One female in the high dose group gave birth to one dead male pup only, leading to a gestation index at this dose level of 90%. This was considered to be incidental.

The mean duration of gestation was similar in all groups.
The mean number of corpora lutea was not considered to have been affected by treatment with the test item.
There were slightly fewer implantation sites per dam (12.2) in the high dose group compared to the control group (14.8). However, this was within the range of the historical control data and was not statistically significant.
Post-implantation loss was statistically significantly increased (3.0 per dam compared to 0.3 in the control group). This was also within the range of the historical control data but it cannot be excluded that this was a test item-related effect. No test item-related findings were noted in the low and mid dose groups on the implantation rate and post-implantation loss.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The organ/body weight ratios of the kidneys, liver and adrenals were slightly increased and the thymus slightly decreased in both males and females of the high dose group. In the mid dose group the organ/body weight ratio of the liver was slightly increased in the males and females. In the low dose group no test item-related findings were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In the high dose group a thickened stomach was noted in 4 males. One male had enlarged adrenal glands. A thickened stomach was noted in 4 females and crateriform retractions in 2 females. The lungs of one female did not collapse at necropsy. This was considered likely to be due to aspiration of the test item. In the mid dose group a thickened stomach was noted in one male, retractions of the stomach were noted in one female. Foci were found on the stomach of one female in the low dose group.

All these macroscopical findings correlated with microscopical findings and were considered to be test item-related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Under the conditions of this study, test item-related changes in the kidneys, urinary bladder, stomach, adrenal glands and bone marrow were observed. In the kidneys, urothelial and collecting duct hyperplasia occurred in high dose group animals along with granular casts and an increased incidence and severity of tubular basophilia and mononuclear cell infiltrates. Minimal granular casts were also found in one mid dose male. Urothelial hyperplasia in the urinary bladder was recorded in one male of the high dose group and in females of the mid and high dose groups.

In the stomach, various degrees of ulceration/erosion, squamous hyperplasia, hyperkeratosis, parakeratosis, pustules, submucosal inflammation and edema were recorded in the forestomach of animals of all dose groups. Mucosal necrosis as well as submucosal edema and inflammation were found randomly distributed in the glandular stomach of mid and high dose group animals.

The histopathologic changes in the kidney and the urinary bladder observed in the mid and high dose groups were consistent with possible irritant effect of the test article and/or its metabolite excreted in the urine. The lesions observed in the forestomach in all dose groups and in the glandular stomach of some individuals in the mid and high dose groups represented a localized stomach reaction to a repeatedly gavaged irritant test material. These changes noted in the kidneys, bladder and stomach were considered adverse.

In addition, there was a slight increase in the incidence and severity of extramedullary hematopoiesis in the adrenal glands (cortices) of females of the high dose group. This was interpreted to be secondary to the inflammatory response in the stomach and possible loss of small quantities of blood through the gastric erosions/ulcers. The slightly increased granulopoiesis in the bone marrow observed in occasional females of the low dose group, and males and females of the mid and high dose groups were interpreted to be secondary to the inflammatory response in the stomach. The effects noted in the adrenals and bone marrow were thus considered adaptive and therefore, not adverse.

Effect levels (P0)

open allclose all
Dose descriptor:
LOEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: localized irritant effects on stomach due to gavage of an irritant
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: localized irritant effects on stomach due to gavage of an irritant
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: clinical signs (salivation), clinical chemistry (increased urea), histopathology (bladder, kidney)
Dose descriptor:
NOEL
Remarks:
reproduction/developmental
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: clinical signs (salivation), clinical chemistry (increased urea), histopathology (bladder, kidney)
Dose descriptor:
LOAEL
Remarks:
reproduction/developmental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: pup weight, litter size, post-implantation loss, postnatal loss

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
In the high dose group, the mean number of pups per litter was statistically significantly reduced (9.2 compared to 14.5 in the control group). This was outside the range of the historical control data. The number of pups per litter in the low and mid dose groups were not considered to have been affected by treatment with the test item.

Postnatal loss was statistically significantly increased (9 in total in 5 litters compared to none in the control group) in the high dose group. In the mid dose group, 3 pups were lost in 2 litters. This was within the range of the historical control data. In the low dose group, no postnatal loss was noted.

CLINICAL SIGNS (OFFSPRING)
No clinical signs were noted at the first litter check or during lactation for any pup in any group

BODY WEIGHT (OFFSPRING)
In the high dose group, the mean body weight of the pups was reduced on day 1 (5.3 grams compared to 5.8 grams in the control group) and was still reduced on day 4 post partum (7.4 grams compared to 8.0 grams in the control group). However, the pups in all groups gained a similar amount of weight over these 4 days. This reduction in body weight was considered to be a test item-related effect.

Mean body weight in the low and mid dose groups was similar to that of the control group.

GROSS PATHOLOGY (OFFSPRING)
In the high dose group, no milk was found in the stomach of 2 female pups in one litter which were already dead at the first litter check. One of these pups had a shortened lower jaw. In another litter, 3 males and 1 female also had no milk in the stomach and were found dead on day 2 post partum. These findings were considered to be incidental.

No other findings were noted at the first litter check or during lactation for any pup in any group.

No abnormal macroscopical findings were noted for any pup in any group at necropsy.

OTHER FINDINGS (OFFSPRING)
On day 4 post partum, the sex ratios were close to 50% in all groups.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: localized irritant effects on stomach due to gavage of an irritant
Dose descriptor:
LOAEL
Remarks:
systemic
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: pup weight

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Conclusion:

Although developmental toxicity effects like reduced pup weight, litter size and increased post-implantation and postnatal loss were observed in animals of the highest dose group, these effects have to be considered as secondary to maternal toxicity. Effects observed in parental animals of the highest dose group include sedation, salivation, and irritation effects in the stomach and the bladder due to the irritating nature of the test substance, confirmed by macroscopic and histopathologic findings in the respective organs. These effects were very likely to explain reduced weight gain and reduced absolute body weights and were already observed in less pronounced form in the animals of the mid dose group.

Considering the characteristics and severity of those adverse maternal findings, the observed developmental effects observed in utero and post-partum have to be considered as non-specific and secondary to maternal toxicity.

Therefore, the test substance does not have to be classifed as toxic to reproduction according to the criteria of the DSD and the CLP-regulation.

Applicant's summary and conclusion