Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28th June to 28th September 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390TX
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent on 22 June 2004.
- Age at study initiation: On arrival, the animals were approximately 6 weeks old.
- Weight at study initiation: A sample of the males were in a weight range of 161-174 g and an equivalent sample of females were in the weight range of 118-130 g.
- Housing:
The animals were initially housed 2 per cage. The cages were made of polypropylene, with stainless steel grid bottoms and mesh tops, and measured ca 42 x 27 x 20 cm. A stainless steel food hopper and a polypropylene or polycarbonate water bottle was provided for each cage. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage. The cages were suspended on racks, each full rack containing 6 rows of 4 cages. Male and female cages were racked separately.
A few days prior to pairing for mating, males were transferred to individual grid-bottomed cages (ca 58 x 38.5 x 20 cm) of a similar design.
Mated females were transferred to individual 42 x 27 x 20 cm solid-bottomed cages. Sterilised wood shavings were provided as bedding in these cages. An analytical certificate for the wood shavings is present within the study report. Just prior to anticipated parturition, some white paper tissue was provided as nesting material.
It was protocolled that at a suitable time following completion of mating, the males were to be rehoused with their original cage-mates. However, after mating the males were retained individually housed. This protocol deviation did not affect the outcome or integrity of the study.
- Diet (e.g. ad libitum):
Rat and Mouse Breeder Diet No. 3 Expanded SQC supplied by Special Diets Services Ltd., Stepfield, Witham, Essex. The diet was available ad libitum. The diet was supplied with a batch analysis for major nutritive components and significant contaminants.
The food was not considered to contain any additional substances in sufficient concentration to have any influence on the outcome of the study.
- Water (e.g. ad libitum):
The animals had access to domestic mains water ad libitum. The water supply is analysed at 6 monthly intervals for dissolved and suspended materials, including a range of significant contaminants.
The water was not considered to contain any additional substances in sufficient concentration to have any influence on the outcome of the study.
- Acclimation period:
The animals were allowed to acclimatise to the Rodent Toxicology Accommodation for approximately 2 weeks between arrival until commencement of treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature remained within the target range of 20°C ± 2°C.
- Humidity (%): Daily monitoring indicated that humidity was out with the target range of 50% ± 15% on four occasions during the study (actual range 48-74%).
- Air changes (per hr): Minimum of 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Light hours were 0700-1900 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared by simple mixing of appropriately weighed quantities of test item to measured volumes of vehicle (water for irrigation).
Vehicle formulation (without the item under investigation) was prepared for the Control animals.
Formulations were prepared and dispensed daily for dosing.


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A Water used for irrigation
- Concentration in vehicle: Individual animal doses were calculated using a daily weighing. A constant dose volume of 20 ml/kg body weight was used.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation:
A few days prior to the initiation of mating, the males were separated into individual grid bottomed cages. Animals were paired on a one male to one female basis, with both animals being in the same treatment group. Each female was transferred to the cage of its appropriate co-group male near the end of the work day, where it remained until mating had occurred.
Each female remained with its first designated male for a maximum of 7 consecutive nights. The protocol provided for re-mating of any females that did not show a positive mating sign, but this was not needed.
- Proof of pregnancy:
Vaginal lavages were taken daily early each morning from the day of pairing until mating had occurred, and the stage of oestrus observed in each lavage was recorded. The day of detection of a copulatory plug in situ and/or sperm in the lavage was designated Day 0 of gestation.
For each female, the time taken to show a positive mating sign and the number of failed opportunities to mate (oestruses passed without a sign of mating) were evaluated.
- After successful mating each pregnant female was caged (how):
Mated females were transferred to individual 42 x 27 x 20 cm solid-bottomed cages. Sterilised wood shavings were provided as bedding in these cages. Just prior to anticipated parturition, some white paper tissue was provided as nesting material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to study commencement, trial formulations of 0.1-200 mg.ml-1 WRS-2390TX were investigated for stability, concentration and homogeneity. This work was carried out the Toxicology Support laboratory under a separate protocol (Inveresk Study No. 421667, Method No. 2166).
Formulations were analysed on 2 occasions during the study treatment period, on Day 1 and during Week 4. The protocol also provided for samples from 2 further occasions to be retained, deep frozen, but not analysed. By oversight, samples from Week 2 were not taken; samples were taken from Week 3 and analysed, but the results were not valid due to poor chromatography. Although this deviated from the protocol, the study was not compromised by these actions.
On each occasion, triplicate samples were withdrawn from each formulation (including Control).

Duration of treatment / exposure:

The males were treated for 4 weeks overall, starting from 2 weeks prior to pairing for mating through to termination.
The females were treated for 2 weeks prior to pairing for mating, then through mating, gestation and until termination on or after Day 4 of lactation.

Frequency of treatment:

The animals were dosed once daily orally by gavage at a dose volume of 20 mL/kg, using a steel cannula. The volume to be administered to each animal was determined on each day by the weight of that animal as measured at the time of administration, except during late gestation; from Day 16 until parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.

Details on study schedule:

nda

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
7.5 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested

Details on study design:

10 males and 10 females (20 in total) per dose group

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
All animals were checked early in the morning and as late as possible each day for viability and at frequent intervals throughout each day for any signs of ill health or reaction to treatment. The onset, intensity and duration of any clinical sign was recorded with particular attention paid to the first 1-2 hours after dosing, then as required thereafter.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
The onset, intensity and duration of any clinical sign was recorded with particular attention paid to the first 1-2 hours after dosing, then as required thereafter.


BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded one week prior to commencement of dosing, then daily to determine the correct dose volume to be administered. For brevity, weekly data are presented for males, and for females during the pre-mating period. Weights for females are additionally presented on Days 0, 7, 14 and 20 of gestation and Days 1, 3 and 4 of lactation.


FOOD CONSUMPTION:
For both sexes, consumption was recorded weekly from one week prior to commencement of treatment through until pairing for mating.
For males, consumption was also recorded for a complete one-week period, after cohabitation with females.
For mated females, consumption was recorded over Days 0-7, 7-14 and 14-20 of gestation, then Days 0-4 of lactation.

Oestrous cyclicity (parental animals):

nda

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Organs fixed:
- Testes (weight recorded individually), fixed in Bouin’s fluid
- Epididymides (weighed individually)
- Seminal vesicles and coagulating gland
- Prostrate gland
- Pituitary gland

A section of each epididymis, together with a transverse section from each testis, was prepared from each male.

From the testes, an additional section was cut, stained with PAS-haematoxylin, and evaluated.

Litter observations:

STANDARDISATION OF LITTERS
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which first pups were born) was designated Day 0 of lactation. The duration of gestation in days was evaluated. The number of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were sexed, counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily until Day 4 of lactation. These pups were weighed en masse, sexes separate, on Days 1 and 4 of lactation.
Deficiencies in lactation and maternal care were recorded.


PARAMETERS EXAMINED
The following parameters were examined in offspring:
All pups, either premature decedents, or those sacrificed at terminal necropsy were examined for any visible abnormalities. Abnormal decedent pups were, where practical, fixed for possible future examination. Externally normal decedents were discarded.
Birth index, live birth index and viability index were calulated.


GROSS EXAMINATION OF DEAD PUPS:
No possible cause of death was not determined for pups born or found dead. Abnormal decedent pups were, where practical, fixed for possible future examination.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The males were killed when mating was completed and all animals had been dosed for 4 weeks.
- Maternal animals: The females were killed on or shortly after Day 4 of lactation. At the high dose, where many pups died shortly after birth, surviving pups were sacrificed prematurely on welfare grounds when it had become apparent that their survival was compromised; some females were also sacrificed prematurely on welfare grounds by similar reasoning.


GROSS NECROPSY
At terminal necropsy, adult animals were killed by carbon dioxide asphyxiation followed by exsanguination.
After sacrifice by exposure to carbon dioxide, a terminal body weight was recorded, followed by exsanguination. These body weights were used for statistical analysis of the male organ weights.
The adult animals were subjected to a gross necropsy, in which the cranial, thoracic and abdominal contents were examined macroscopically; any findings were recorded and representative samples of abnormal tissues were taken and fixed in neutral buffered 10% formalin as appropriate.

The following organs were also fixed:
Ovaries
Uterus, cervix and vagina
Testes (weight recorded individually), fixed in Bouin’s fluid
Epididymides (weighed individually)
Seminal vesicles and coagulating gland
Prostrate gland
Pituitary gland

Paired organs were weighed separately but the sum of the individual organs has been used for reporting purposes.
The reproductive tract of all females were examined for signs of implantation; the number of implantation sites was recorded.
The adult carcasses were discarded following examination.


HISTOPATHOLOGY / ORGAN WEIGHTS
A section of each epididymis, together with a transverse section from each testis, was prepared from each male. A section from each ovary was prepared from each Control and High dose female. Sections were cut 4-6μm thick, stained with haematoxylin and eosin (H&E) and evaluated by a pathologist.
From the testes, an additional section was cut, stained with PAS-haematoxylin, and evaluated. No histological examination was conducted on the other preserved tissues and organs.

Postmortem examinations (offspring):

SACRIFICE
- The offspring were sacrificed on or shortly after day 4 of lactation. At the high dose, where many pups died shortly after birth, surviving pups were sacrificed prematurely on welfare grounds when it had become apparent that their survival was compromised.
Pups were killed by injection of sodium pentobarbitone.


NECROPSY
All pups, either premature decedents, or those sacrificed at terminal necropsy were examined for any visible abnormalities. Abnormal decedent pups were, where practical, fixed for possible future examination. Externally normal decedents were discarded.

Statistics:

Body weight and food consumption of males, and of females prior to mating were analysed by analysis of variance or the Kruskal-Wallis non-parametric analysis as appropriate.
Organ weight data were analysed by analysis of variance and analysis of covariance using the terminal body weight as the single covariate.
Histology data were analysed by Fisher’s Exact Probability Test.
All statistical tests were 2-sided, conducted at the 5% significance level, and pairwise comparisons were only made against the Control group.
For other parameters, interpretation was made by inspection of the individual and group values.

Reproductive indices:

Reproductive indices calculated:
- Fertility index (male)
- Fertility index (female)
- Gestation index

Offspring viability indices:

Offspring indices calculated:
- Birth index
- Live birth index
- Viability index

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:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At 50 mg/kg/day, most animals showed piloerection and excessive salivation; both of these signs were observed intermittently. Animal 77 showed rolling gait, hunched posture, staining on the abdomen at or shortly after littering; Animal 78 had hunched posture at this time: these findings may have been associated with the littering process.
The other findings at 50 mg/kg/day, and all findings at the lower dose levels, were not considered to be related to treatment. There were no premature decedents throughout the study.


BODY WEIGHT (PARENTAL ANIMALS)
Among males treated at 7.5 and 50 mg/kg/day, there was a reduction in weight gain during the first week of treatment. Weight gain thereafter was similar to Control. Weight gain of males at 2 mg/kg/day was similar to Control.
Among females treated at 7.5 and 50 mg/kg/day, there was a dose-related reduction in body weight gain over the first week of treatment. At Day 7, the absolute body weight at 50 mg/kg/day was significantly lower than Control. Over the second week of treatment, weight gain at 50 mg/kg/day was greater than Control, such that by Day 14 of treatment, overall weight gain and absolute body weight were only slightly lower than Control. At 7.5 mg/kg/day, however, weight gain over the second week was also lower than Control, such that the overall weight gain to Day 14 of treatment and absolute body weight at Day 14 were significantly lower than Control. Weight gain of females at 2 mg/kg/day during the pre-mating period was similar to Control.
During gestation, mean weight gains at 7.5 and 50 mg/kg/day were lower than Control; slight differences in weight gains at 2 mg/kg/day were not considered to have been related to treatment.
Because all litters at 50 mg/kg/day were lost, it was not appropriate to compare body weights during lactation with the Control. At 2 and 7.5 mg/kg/day, body weight performance during lactation did not provide any indication of an adverse effect of treatment.


FOOD CONSUMPTION (PARENTAL ANIMALS)
Males treated at 50 mg/kg/day showed a slight but significant reduction in food consumption during the first week; thereafter, consumption at this level was similar to Control. At 7.5 mg/kg/day, consumption during the second week was marginally lower than Control, although the difference was not statistically significant. Food consumption by males at 2 mg/kg/day was similar to Control.
Among females treated at 7.5 and 50 mg/kg/day, there was a dose-related reduction in food consumption in the first week of treatment. During the second week of treatment, consumption by females at 50 mg/kg/day was only marginally lower than Control, but consumption at 7.5 mg/kg/day was significantly lower. Food consumption by females at 2 mg/kg/day during the pre-mating period was similar to Control.
Mean food consumption during gestation at 7.5 and 50 mg/kg/day, and over Days 0-4 of lactation at 7.5 mg/kg/day, was lower than Control; consumption at 2 mg/kg/day during gestation and lactation was similar to Control.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
n/a


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
nda


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
nda


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
At 50 mg/kg/day, there was a slight increase in the number of nights to positive mating sign, although 9/10 pairs mated within 4 nights, i.e. within the first oestrous cycle. Only 8/10 females became pregnant at this level.
Mating performance and Fertility Indices at the lower levels were similar to Control.
The mean duration of gestation at 50 mg/kg/day was slightly increased, with no female showing a 21 day duration. The duration of gestation at the lower levels was similar to Control.


ORGAN WEIGHTS (PARENTAL ANIMALS)
At 50 mg/kg/day, 6/10 males had testes weights that were markedly lower than Control; the testes weights of the remaining 4 males were similar to Control. The resultant group mean weight was significantly lower. Mean epididymides weights at this level were also significantly lower than Control; the animals that had low testes weights generally also had low epididymides weights.
Mean testes and epididymides weights at 2 and 7.5 mg/kg/day were similar to Control.


GROSS PATHOLOGY/HISTOPATHOLOGY (PARENTAL ANIMALS)
At 50 mg/kg/day small testes were observed in 6/10 males, with 3 of these animals having flaccid testes and 1 with small epididymides.
At 50 mg/kg/day severe seminiferous epithelial degeneration was observed in 6/10 males, which correlated with the small, flaccid testes seen at necropsy. These animals also showed mild testicular interstitial cell hyperplasia. There was mild to marked oligospermia and moderate to marked sloughing of spermatogenic cells in the epididymides from the same animals, which in one animal correlated with small epididymides at necropsy.
At 7.5 mg/kg/day minimal sloughing of spermatogenic cells was also present in the epididymides of one male. This was not considered to provide sufficient evidence of an effect of treatment, as this finding could have occurred spontaneously.
There were no notable findings in the testes and epididymides at 2 mg/kg/day, or in the ovaries at 50 mg/kg/day.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
At 50 mg/kg/day, the number of implants was lower than Control, with a corresponding reduction in the number of pups born. Within 1-2 days of birth, marked pup mortality became apparent and for welfare considerations all surviving pups at this level were sacrificed.
At 7.5 mg/kg/day, the number of implants, and therefore of pups born, was reduced but pup survival was not affected.
At 2 mg/kg/day, slight intergroup differences in litter size and survival were not considered to be related to treatment.


CLINICAL SIGNS (OFFSPRING)
There were no obvious abnormalities among the pups, except for one pup in Litter 71 with an undefined abnormality; this single finding, although not correctly described, could not be attributed to treatment.
Occasional minor findings, such as minor bruising, cold, that are typical among pre-weanlings were noted. These have not been tabulated but are being retained in the study data.


BODY WEIGHT (OFFSPRING)
At 7.5 mg/kg/day, mean pup weights on Day 1 were slightly lower than Control, and the gain to Day 4 was also lower than in the Control. Litter weights reflected the differences in litter size and pup weight.
At 2 mg/kg/day, there were no obvious effects on litter and pup weights.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Toxicity of WRS-2390TX was indicated at 7.5 and 50 mg/kg/day by decreases in body weight gain and food consumption, and at 50 mg/kg/day also by piloerection and salivation.

At 50 mg/kg/day, 6/10 males had low testes weights; these testes had severe seminiferous epithelial degeneration and mild interstitial cell hyperplasia. There was mild to marked oligospermia and moderate to marked sloughing of spermatogenic cells in the epididymides of these animals; group mean epididymides weight at this level was lower than Control. At 50 mg/kg/day, 2/10 pairs failed to mate; both males had low testes weights.

The mean duration of gestation at 50 mg/kg/day was slightly increased.

The mean number of implants at 50 mg/kg/day was lower than Control, and pup mortality markedly increased, such that surviving pups at this level were sacrificed for welfare considerations.

At 7.5 mg/kg/day, reproductive effects were confined to a reduced number of implants, and therefore of pups born, and slight reduction in mean litter and pup weights.

Under the conditions of the study, the No Observed Effect Level (NOEL) for both sexes of adults, and for reproductive effects, was 2 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the No Observed Effect Level (NOEL) for both sexes of adults, and for reproductive effects, was 2 mg/kg/day. The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability & repeatability).

Executive summary:

In a Reproduction/Developmental Toxicity Screening Test in Rats (Inveresk report number: 24474) the No Adverse Effect Level (NOAEL) for both sexes was considered to be 2 mg/kg/day for the test material.

Toxicity of WRS-2390TX was indicated at 7.5 and 50 mg/kg/day by decreases in body weight gain and food consumption, and at 50 mg/kg/day also by piloerection and salivation.

At 50 mg/kg/day, 6/10 males had low testes weights; these testes had severe seminiferous epithelial degeneration and mild interstitial cell hyperplasia. There was mild to marked oligospermia and moderate to marked sloughing of spermatogenic cells in the epididymides of these animals; group mean epididymides weight at this level was lower than Control. At 50 mg/kg/day, 2/10 pairs failed to mate; both males had low testes weights.

The mean duration of gestation at 50 mg/kg/day was slightly increased.

The mean number of implants at 50 mg/kg/day was lower than Control, and pup mortality markedly increased, such that surviving pups at this level were sacrificed for welfare considerations.

At 7.5 mg/kg/day, reproductive effects were confined to a reduced number of implants, and therefore of pups born, and slight reduction in mean litter and pup weights.

The method was determined according to OECD Guideline 421 and is in compliance with the OECD principles of Good Laboratory Practice.