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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 28th to April 12th, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
reproductive toxicity, other
Remarks:
Data regarding reproductive toxicity obtained from the prenatal developmental toxicity study (see section 7.8.2 of this IUCLID dossier)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From March 28th to April 12th, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: Data regarding reproductive toxicity obtained from the prenatal developmental toxicity study conducted according to OECD414 version May 1981 (see section 7.8.2 of this IUCLID dossier)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl: CD®BR VAF/Plus strain
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent.
- Age at study initiation: 8 - 10 weeks old.
- Weight at study initiation: weight range of 178 - 230 g.
- Housing: the animals were housed five to a cage in suspended stainless steel cages equipped with solid sides and wire grid front, back, floor and top. The cages constituting each treatment group were dispersed within the batteries so that possible environmental influences arising from their spatial distribution were equilibrated as far as possible for all treatments. Throughout the study each cage was identified by a label coloured according to the group and recording the study schedule number, animal numbers, details of treatment and the names of the Study Supervisor and Study Director.
- Diet (e.g. ad libitum): free access to SDS Laboratory Animal Diet No. 1 (pelleted). Results of the regular chemical analyses of food and water are lodged in the laboratory archives.
- Water (e.g. ad libitum): free access to tap water .Results of the regular chemical analyses of food and water are lodged in the laboratory archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22 °C
- Humidity (%): 31-58% relative humidity
- Photoperiod (hrs dark / hrs light): artificial lighting was controlled to give 12 hours light (0800 to 2000 hours) and 12 hours dark per 24 hours.
Route of administration:
oral: gavage
Vehicle:
other: 1 % w/v aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the dosing formulations were stated to be stable for 4 hours at room temperature in the dark and suspensions were therefore prepared daily and dosed on the day of preparation. A series of formulations was prepared by grinding an appropriate amount of the test substance with a small amount of vehicle (1 % methylcellulose) using a mortar and pestle until a smooth paste was formed. The formulation was then made up to the volume required and mixed using a high shear homogeniser. The concentrations used were chosen to give a constant dose volume of 10 ml/kg bodyweight. The test substance was administered as a suspension in 1% methylcellulose. The formulations were stirred using a magnetic stirrer for the duration of the dosing procedure. Control animals received the vehicle alone at the same dose volume. The animals were dosed at approximately the same time each day, where possible, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.

VEHICLE:
- Concentration in vehicle: 0, 1.0, 5.0 and 10.0 mg/ml of Chlorphenesin in the vehicle (1% w/v aqueous methylcellulose).
Details on mating procedure:
A total of one hundred and eleven sexually mature female rats which were time mated to identified males of the same strain was received from Charles River UK Limited, Manston Road, Margate, Kent. The first batch (A) consisted of 66 animals followed by a second batch (B) of 45 animals mated one day later. The day of mating , as judged by the appearance of sperm in the vaginal smear or the presence of a vaginal plug, was considered as Day 0 of pregnancy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of treatment, satisfactory homogeneity and stability data for the formulations under the conditions of use were generated by the Sponsor. Samples of dose formulations prepared for use on the first day of dosing were taken and analysed by analytical department.
A representative sample (approximately 1 ml) of test formulation was accurately weighed and dissolved in a suitable volume (approximately 30 ml) of mobile phase Methanol/water (55/45 v/v). The extract was diluted to volume (50 ml) and appropriately diluted using mobile phase, to provide a solution containing chlorphenesin in the expected concentration range 4 - 8 µg/mI.
The final solution was filtered (Whatman GF/F) and the concentration of chlorphenesin was quantified by high performance liquid chromatography using ultraviolet detection.
Analytical column: Jones Chromatography Ltd, Apex ODS, 5 µm, 250 x 4.6 mm ID.
Mobile phase: Methanol/water (55/45 v/v).
Detector wavelength: UV, 279 nm.

LIMIT OF DETECTION
The limit of detection, defined as the concentration of chlorphenesin in control matrix producing a peak response equivalent to 3 x baseline noise, was determined as 0.075 mg/ml.

DETERMINATION OF CONCENTRATIONS OF CHLORPHENESIN IN DOSE FORMULATIONS PREPARED FOR DAY 1 OF DOSING
Representative samples (approximately 20 ml) of freshly prepared dose formulations were thoroughly mixed by vigorous shaking and duplicate sub-samples (1 ml) were analysed in accordance with the analytical procedure.

VALIDATION OF THE METHOD OF ANALYSIS
On Day 1 of dosing, a procedural recovery was prepared at each inclusion level by fortifying a sample (1 ml) of control vehicle with a solution of chlorphenesin in mobile phase. The method was validated by analysing the procedural recoveries concurrently with the dose formulations in accordance with the analytical procedure.

RESULTS
The analytical results confirm that the doses were accurately formulated for Day 1 of dosing.
Duration of treatment / exposure:
The test substance was administered to time-mated female rats, once per day, from Days 6 to 15 post coitum.
Frequency of treatment:
Daily
Details on study schedule:
A total of one hundred and eleven sexually mature female rats which were time mated to identified males of the same strain was received from Charles River UK Limited, Manston Road, Margate, Kent. The first batch (A) consisted of 66 animals followed by a second batch (B) of 45 animals mated one day 1. The day of mating , as judged by the appearance of sperm in the vaginal smear or the presence of a vaginal plug, was considered as Day 0 of pregnancy. The mated female rats arrived at laboratory one day later after mating and the treatment occurred from Days 6 to 15 post coitum.
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
15 female rats for batch A and 10 female rats for batch B for each dose (total 25 female rats x 4 dose = 100 female rats used for the test).
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: the dosages of 0 (Control), 10, 50 and 100 mg/kg bw/day were selected with reference to a 28 day study in non-pregnant rats performed at these laboratories (See section 7.5.1 Repeated dose toxicity:oral of this IUCLID dossier) in which the highest dosage (1000 mg/kg bw/day) produced clear evidence of toxicity manifested principally as reduced bodyweight gain and food intake, and increased water consumption. Increased water consumption was also noted in males receiving the intermediate dosage level (100 mg/kg bw/day), as well as minor effects on haemoglobin and plasma albumin levels. The low dose level (10 mg/kg bw/day) was determined as the `No Adverse Effect Level'.

- Rationale for animal assignment (if not random): allocation to treatment groups occurred on Day 2 post coitum when animals were assigned to four groups by computerised stratified randomisation on the basis of bodyweight in order to give approximately equal initial group mean bodyweights within each batch. Adjustments were made to the group allocation in order to ensure an acceptable distribution of the males to which females were mated. Following allocation, the animals were earmarked to give individual identification. Prior to the commencement of treatment, a review of animal health was undertaken by a veterinary officer. Spare rats not allocated to the study were removed after the start of treatment.
Positive control:
Not present.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily. All animals were regularly handled and observed daily for obvious changes and for signs of reaction to treatment or ill health/moribundity.
- Cage side observations: recorded in study Daybook

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily.

BODY WEIGHT:
- Time schedule for examinations: all animals were weighed initially on arrival (= Day 1 post coitum for Batch A; Day 2 for Batch B) and on Days (2 Batch A), 3, 6, 8, 10, 12, 14, 16, 18 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE :food consumption was measured from weighday to weighday commencing on Day 3 post coitum. Food consumption was determined as cage mean values and as group mean values. The substance was administrated by oral gavage and not in food.

WATER CONSUMPTION AND COMPOUND INTAKE: water consumption measurement was not instituted in the absence of an apparent effect identified by visual appraisal. The substance was administrated by oral gavage and not in drinking water

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 20.On Day 20, the animals were killed by CO2 asphyxiation, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs. Abnormal tissues were preserved at the discretion of the post mortem pathologist.
- Organs examined: maternal organs include ovaries and uteri.
Oestrous cyclicity (parental animals):
The ovaries and uteri were examined immediately to determine: number of corpora lutea, number and distribution of live foetuses, number and distribution of embryofoetal deaths, individual foetal weight from which the litter weight was calculated, gross foetal abnormalities.
Sperm parameters (parental animals):
Not expected: only premated female rats were tested.
Litter observations:
Embryofoetal deaths were classified as:
- Early: only placental remnants visible at termination.
- Late: both placental and embryonic remnants visible at termination.
Uteri or individual uterine horns without visible implantations were examined for evidence of implantation using a modified Salewski technique (Salewski, 1964).
Live foetuses were examined externally and weighed. Half the foetuses in each litter were preserved in Bouin's solution for subsequent free-hand sectioning to discover visceral abnormalities (Wilson technique (Wilson, 1965)); the remainder were fixed in 74 OP industrial methylated spirit for subsequent macroscopic examination, evisceration, clearing and alizarin staining (modified Dawson technique (Dawson, 1926)) for skeletal examination. Foetuses showing suspected abnormalities were processed by the more appropriate technique; tissues from affected foetuses were processed into 56°C MP paraffin wax, sectioned at 5 gm and stained with haematoxylin and eosin and examined histologically for clarification of initial. Photographic records of these abnormalities were made either at gross, visceral or skeletal examination, as appropriate, and included a normal litter mate at the discretion of the teratologist.
All foetuses were uniquely identified to allow correlation of initial with subsequent findings and were sexed by gonadal inspection following preservation.
Structural changes are presented as:
- Malformations: rare and/or probably lethal, e.g. exencephaly, anury.
- Anomalies: minor differences from 'normal' that are detected relatively frequently either by free-hand sectioning, e.g. increased renal pelvic dilatation, or at skeletal examination, e.g. bipartite centrum.
- Variants: alternative structures occurring regularly in the control population, e.g. unossified sternebra(e).
Postmortem examinations (parental animals):
On Day 20, the animals were killed by CO2 asphyxiation, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs. Abnormal tissues were preserved at the discretion of the post mortem pathologist. The ovaries and uteri were examined immediately to determine: number of corpora lutea, number and distribution of live foetuses, number and distribution of embryofoetal deaths, individual foetal weight from which the litter weight was calculated and gross foetal abnormalities.
Postmortem examinations (offspring):
Not performed. All female rats were killed before the birth of the offsprings.
Statistics:
Significance tests, employing analysis of variance followed by comparison of treated groups with controls, were performed on the following parameters and results are presented in relevant tables of this report: bodyweight change, food consumption, litter data and foetal abnormalities.
Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance (Snedecor and Cochran, 1967) followed by Williams' test (Williams, 1971/2)) or non-parametric tests (Kruskal-Wallis (Hollander and Wolfe, 1973) followed by Shirley's test (Shirley, 1977)) were used to analyse these data, as appropriate. Data relating to food consumption were analysed on a cage basis; for bodyweight change the analysis used the individual animal as the basic experimental unit. For litter data, the basic sample unit was the litter and, due to the preponderance of non-normal distributions, non-parametric analyses were routinely used. Analysis of incidence of foetal malformations and anomalies was performed using a one-tailed permutation test (Edgington, 1980).
Where 75% or more of the values for a given variable were the same, the Fisher's exact test (Fisher, 1950) was used.
Reproductive indices:
# Individual litter values
In assessing litter parameters, pre-implantation loss was calculated as a percentage from the formula:
(No. of corpora lutea - no. of implantations)/ No. of corpora lutea x 100
Post implantation loss was similarly calculated from the formula:
(No. of implantations - no. of live foetuses)/ No. of implantations x 100
Litter weight and mean foetal weight were calculated from individual foetal weight. Sex ratio was expressed as the percentage of males per litter.

# Group litter values
Group mean values calculated from individual litter values were presented including valid data from all animals with live foetuses at termination (Day 20 of pregnancy).
All derived values (eg percentages, ratios) were first calculated within each litter and the group value derived as a mean of the individual litter values.

Specific reproductive indices: e.g. number of non pregnant rats, pre-implantation loss, post-implantation loss, ecc
Offspring viability indices:
# Individual litter values
In assessing litter parameters, pre-implantation loss was calculated as a percentage from the formula:
(No. of corpora lutea - no. of implantations)/ No. of corpora lutea x 100
Post implantation loss was similarly calculated from the formula:
(No. of implantations - no. of live foetuses)/ No. of implantations x 100
Litter weight and mean foetal weight were calculated from individual foetal weight. Sex ratio was expressed as the percentage of males per litter.

# Group litter values
Group mean values calculated from individual litter values were presented including valid data from all animals with live foetuses at termination (Day 20 of pregnancy).
All derived values (eg percentages, ratios) were first calculated within each litter and the group value derived as a mean of the individual litter values.

Specific for litter: e.g. in utero deaths, no. of malformation (visceral and skeletral), ecc
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A transient increase in post dose salivation was observed in 4/25 animals treated at 100 mg/kg/day. This sign was noted in each animal on one occasion from Day 11 to 15 post coitum. Increased salivation was not noted at 10 or 50 mg/kg/day. Fur loss was also noted from Day 8 post coitum in 9, 13, 13 and 16/25 animals treated at 0, 10, 50 and 100 mg/kg/day respectively.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight gain showed no effect of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption measurement did not reveal any effect of treatment. The substance was administrated by oral gavage and not in food.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Some data regarding estrous cycle could be obtained based on the number of non-pregnant rats and on the ratio No. of implantation/ No. of corpora lutea for each dose group compared with control group. In fact the number of non pregnant rats in control group was comparable to the number of non pregnant animal in the treated groups (non pregnant animals on total 25 rats for group: 2, 2, 0 and 1 respectively for control, 10 mg/kg/day, 50 mg/kg/day and 100 mg/kg/day group). Also the ratio No. of implantation/ No of corpora lutea of control group was comparable with to the ratio in the treated groups (ratio as mean values for each group: 12.8/13.8, 12.8/13.4, 3.0/13.8 and 12.3/13.5 respectively for control, 10 mg/kg bw/day, 50 mg/kg bw/day and 100 mg/kg bw/day group).
Reproductive function: sperm measures:
not specified
Description (incidence and severity):
The female rats arrived at laboratory after mating.
Reproductive performance:
no effects observed
Description (incidence and severity):
Some data regarding reproductive performance could be obtained based on the number of non-pregnant rats and on the ratio No. of implantation/ No. of corpora lutea for each dose group compared with control group. In fact the number of non pregnant rats in control group was comparable to the number of non pregnant animal in the treated groups (non pregnant animals on total 25 rats for group: 2, 2, 0 and 1 respectively for control, 10 mg/kg/day, 50 mg/kg/day and 100 mg/kg/day group). Also the ratio No. of implantation/ No of corpora lutea of control group was comparable with to the ratio in the treated groups (ratio as mean values for each group: 12.8/13.8, 12.8/13.4, 13.0/13.8 and 12.3/13.5 respectively for control, 10 mg/kg bw/day, 50 mg/kg bw/day and 100 mg/kg bw/day group).
Dose descriptor:
NOEL
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: based on the number of non-pregnant rats and on the ratio No. of implantation/ No. of corpora lutea for each dose group compared with control group.
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
All female rats were killed before the birth of the offsprings. The data regarding foetal examination were reported. It was considered that there was no effect on litter values. Marginal differences between the controls and animals receiving 100 mg/kg bw/day seen for implantation rate, embryonic deaths and resulting number of live young were not statistically significant and are considered not to be of toxicological importance, particularly in the absence of a toxic effect to the dam at this dosage level.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
All female rats were killed before the birth of the offsprings. The data regarding foetal examination were reported. The number and distribution of malformations, visceral anomalies and skeletal variants, together with the absence of statistically significant differences indicated that there was no treatment-related effect of chlorphenesin on these categories of abnormality.
For skeletal anomalies, the distribution amongst litters was higher at all dosage levels than for controls, attaining statistical significance at 50 mg/kg bw/day. However, in the absence of a clear dosage-relationship or of an obvious trend in any specific type of skeletal anomaly, these differences were considered to be unrelated to treatment.
Key result
Dose descriptor:
NOEL
Generation:
other: based on foetal examination
Effect level:
ca. 100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: See remarks
Reproductive effects observed:
no
Conclusions:
NOEL Chlorphenesin (prenatal developmental toxicity in rats) = 100 mg/kg bw/day. Based on the data regarding the number of pregnant rats and on the ratio No. of implantation/ No. of corpora lutea for each dose group compared with control group this NOEL= 100 mg/kg bw/day should be applied also for reproductive toxicity.
Executive summary:

Chlorphenesin has been tested to evaluate the effect of on pregnancy and in utero development of the rat. Doses of 0 (Control), 10, 50 and 100 mg/kg were administered to groups of twenty-five time-mated females, by oral gavage once per day, from Days 6 to 15 post coitum inclusive. Clinical signs, bodyweight and food consumption data were collected. On Day 20 all females were killed and subjected to post mortem examination; litter values were determined and foetuses preserved for subsequent examination of visceral and skeletal changes.

 

The following comments in relation to principal findings during the study are made in summary:

- all dosages were without adverse effect on embryofoetal survival, growth and development in utero;

- there was no evidence of maternal toxicity. Although there was an apparent increase in the incidence of fur loss noted in all treated groups during the live phase of the study, post mortem examination indicated that there was an increase only at the high dosage level, and that the fur loss was mostly classified as minimal in degree and sometimes associated with scabbing. It is therefore considered unlikely that this fur loss was associated with the administration of test item;

- 100 mg/kg/day was associated with transient post dosing salivation for 4/25 females on isolated occasions towards the end of the treatment period;

- maternal bodyweight gain and food intake were not affected at any dosage.

 

In conclusion, treatment of the parent female produced no selective effect on embryofoetal development.

The maximum no observable effect level for selective toxicity to the developing foetus was therefore considered to be 100 mg/kg bw/day. There was no evidence of maternal toxicity. Based on the data regarding the number of pregnant rats and on the ratio No. of implantation/ No. of corpora lutea for each dose group compared with control group this NOEL= 100 mg/kg bw /day should be applied also for reproductive toxicity.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 1981
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Chlorphenesin
EC Number:
203-192-6
EC Name:
Chlorphenesin
Cas Number:
104-29-0
Molecular formula:
C9H11ClO3
IUPAC Name:
3-(4-chlorophenoxy)propane-1,2-diol
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
other: Crl: CD®BR VAF/Plus strain
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent.
- Age at study initiation: 8 - 10 weeks old.
- Weight at study initiation: weight range of 178 - 230 g.
- Housing: the animals were housed five to a cage in suspended stainless steel cages equipped with solid sides and wire grid front, back, floor and top. The cages constituting each treatment group were dispersed within the batteries so that possible environmental influences arising from their spatial distribution were equilibrated as far as possible for all treatments. Throughout the study each cage was identified by a label coloured according to the group and recording the study schedule number, animal numbers, details of treatment and the names of the Study Supervisor and Study Director.
- Diet (e.g. ad libitum): free access to SDS Laboratory Animal Diet No. 1 (pelleted). Results of the regular chemical analyses of food and water are lodged in the laboratory archives.
- Water (e.g. ad libitum): free access to tap water .Results of the regular chemical analyses of food and water are lodged in the laboratory archives.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22 °C
- Humidity (%): 31-58% relative humidity
- Photoperiod (hrs dark / hrs light): artificial lighting was controlled to give 12 hours light (800 to 2000 hours) and 12 hours dark per 24 hours.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 % w/v aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the dosing formulations were stated to be stable for 4 hours at room temperature in the dark and suspensions were therefore prepared daily and dosed on the day of preparation. A series of formulations was prepared by grinding an appropriate amount of the test substance with a small amount of vehicle (1 % methylcellulose) using a mortar and pestle until a smooth paste was formed. The formulation was then made up to the volume required and mixed using a high shear homogeniser. The concentrations used were chosen to give a constant dose volume of 10 ml/kg bodyweight. The test substance was administered as a suspension in 1% methylcellulose. The formulations were stirred using a magnetic stirrer for the duration of the dosing procedure. Control animals received the vehicle alone at the same dose volume. The animals were dosed at approximately the same time each day, where possible, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.

VEHICLE:
- Concentration in vehicle: 0, 1.0, 5.0 and 10.0 mg/ml of Chlorphenesin in the vehicle (1% w/v aqueous methylcellulose).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of treatment, satisfactory homogeneity and stability data for the formulations under the conditions of use were generated by the Sponsor. Samples of dose formulations prepared for use on the first day of dosing were taken and analysed by analytical department for accuracy of preparation.
A representative sample (approximately 1 ml) of test formulation was accurately weighed and dissolved in a suitable volume (approximately 30 ml) of mobile phase Methanol/water (55/45 v/v). The extract was diluted to volume (50 ml) and appropriately diluted using mobile phase, to provide a solution containing chlorphenesin in the expected concentration range 4 - 8 µg/mI.
The final solution was filtered (Whatman GF/F) and the concentration of chlorphenesin was quantified by high performance liquid chromatography using ultraviolet detection.
Analytical column: Jones Chromatography Ltd, Apex ODS, 5 µm, 250 x 4.6 mm ID.
Mobile phase: Methanol/water (55/45 v/v).
Detector wavelength: UV, 279 nm.

LIMIT OF DETECTION
The limit of detection, defined as the concentration of chlorphenesin in control matrix producing a peak response equivalent to 3 x baseline noise, was determined as 0.075 mg/ml.

DETERMINATION OF CONCENTRATIONS OF CHLORPHENESIN IN DOSE FORMULATIONS PREPARED FOR DAY 1 OF DOSING
Representative samples (approximately 20 ml) of freshly prepared dose formulations were thoroughly mixed by vigorous shaking and duplicate sub-samples (1 ml) were analysed in accordance with the analytical procedure.

VALIDATION OF THE METHOD OF ANALYSIS
On Day 1 of dosing, a procedural recovery was prepared at each inclusion level by fortifying a sample (1 ml) of control vehicle with a solution of chlorphenesin in mobile phase. The method was validated by analysing the procedural recoveries concurrently with the dose formulations in accordance with the analytical procedure.

RESULTS
The analytical results confirm that the doses were accurately formulated for Day 1 of dosing.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant. A total of one hundred and eleven sexually mature female rats which were time mated to identified males of the same strain was received from Charles River UK Limited, Manston Road, Margate, Kent. The first batch (A) consisted of 66 animals followed by a second batch (B) of 45 animals mated one day later.
- Verification of same strain and source of both sexes: the males and females belonged to the same strain
- Proof of pregnancy: the appearance of sperm in the vaginal smear or the presence of a vaginal plug was considered as Day 0 of pregnancy.
Duration of treatment / exposure:
The test substance was administered to time-mated female rats, once per day, from Days 6 to 15 post coitum.
Frequency of treatment:
Daily
Duration of test:
Oral gavage from days 6 to 15 post coitum inclusive.On day 20 all females were killed and subjected to post mortem examination.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
15 female rats for batch A and 10 female rats for batch B for each dose (total 25 female rats x 4 dose = 100 female rats used for the test).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dosages of 0 (Control), 10, 50 and 100 mg/kg/day were selected with reference to a 28 day study in non-pregnant rats performed at these laboratories (See section 7.5.1 Repeated dose toxicity:oral of this IUCLID dossier) in which the highest dosage (1000 mg/kg/day) produced clear evidence of toxicity manifested principally as reduced bodyweight gain and food intake, and increased water consumption. Increased water consumption was also noted in males receiving the intermediate dosage level (100 mg/kg/day), as well as minor effects on haemoglobin and plasma albumin levels. The low dose level (10 mg/kg/day) was determined as the `No Adverse Effect Level'.

- Rationale for animal assignment (if not random): allocation to treatment groups occurred on Day 2 post coitum when animals were assigned to four groups by computerised stratified randomisation on the basis of bodyweight in order to give approximately equal initial group mean bodyweights within each batch. Adjustments were made to the group allocation in order to ensure an acceptable distribution of the males to which females were mated. Following allocation, the animals were earmarked to give individual identification. Prior to the commencement of treatment, a review of animal health was undertaken by a veterinary officer. Spare rats not allocated to the study were removed after the start of treatment.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily. All animals were regularly handled and observed daily for obvious changes and for signs of reaction to treatment or ill health/moribundity.
- Cage side observations: recorded in study Daybook

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily.

BODY WEIGHT:
- Time schedule for examinations: all animals were weighed initially on arrival (= Day 1 post coitum for Batch A; Day 2 for Batch B) and on Days (2 Batch A), 3, 6, 8, 10, 12, 14, 16, 18 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE : food consumption was measured from weighday to weighday commencing on Day 3 post coitum. Food consumption was determined as cage mean values and as group mean values. The substance was administrated by oral gavage and not in food.

WATER CONSUMPTION AND COMPOUND INTAKE: water consumption measurement was not instituted in the absence of an apparent effect identified by visual appraisal. The substance was administrated by oral gavage and not in drinking water

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 20.On Day 20, the animals were killed by CO2 asphyxiation, dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs. Abnormal tissues were preserved at the discretion of the post mortem pathologist.
- Organs examined: maternal organs include ovaries and uteri.

Ovaries and uterine content:
The ovaries and uteri were examined immediately to determine: number of corpora lutea, number and distribution of live foetuses, number and distribution of embryofoetal deaths, individual foetal weight from which the litter weight was calculated, gross foetal abnormalities.
Embryofoetal deaths were classified as:
- Early: only placental remnants visible at termination.
- Late: both placenta and embryonic remnants visible at termination.
Fetal examinations:
- External examinations: live foetuses were examined externally and weighed. Half the foetuses in each litter were preserved in Bouin's solution for subsequent free-hand sectioning to discover visceral abnormalities (Wilson technique (Wilson, 1965)); the remainder were fixed in 74 OP industrial methylated spirit for subsequent macroscopic examination, evisceration, clearing and alizarin staining (modified Dawson technique (Dawson, 1926)) for skeletal examination. Foetuses showing suspected abnormalities were processed by the more appropriate technique.
- Soft tissue examinations: tissues from affected foetuses were processed into 56 °C MP paraffin wax, sectioned at 5µm and stained with haematoxylin and eosin and examined histologically for clarification of initial observations.
- Skeletal examinations :Photographic records of abnormalities were made either at gross, visceral or skeletal examination, as appropriate, and included a normal litter mate at the discretion of the teratologist.
- Other: structural changes are presented as:
Malformations: rare and/or probably lethal, e.g. exencephaly, anury.
Anomalies: minor differences from `normal' that are detected relatively frequently either by free-hand sectioning, e.g. increased renal pelvic dilatation, or at skeletal examination, e.g. bipartite centrum.
Variants: alternative structures occurring regularly in the control population, e.g. unossified sternebra(e).
Statistics:
Significance tests, employing analysis of variance followed by comparison of treated groups with controls, were performed on the following parameters and results are presented in relevant tables of this report: bodyweight change, food consumption, litter data and foetal abnormalities.
Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance (Snedecor and Cochran, 1967) followed by Williams' test (Williams, 1971/2)) or non-parametric tests (Kruskal-Wallis (Hollander and Wolfe, 1973) followed by Shirley's test (Shirley, 1977)) were used to analyse these data, as appropriate. Data relating to food consumption were analysed on a cage basis; for bodyweight change the analysis used the individual animal as the basic experimental unit. For litter data, the basic sample unit was the litter and, due to the preponderance of non-normal distributions, non-parametric analyses were routinely used. Analysis of incidence of foetal malformations and anomalies was performed using a one-tailed permutation test (Edgington, 1980).
Where 75% or more of the values for a given variable were the same, the Fisher's exact test (Fisher, 1950) was used.
Indices:
# Individual litter values
In assessing litter parameters, pre-implantation loss was calculated as a percentage from the formula:
(No. of corpora lutea - no. of implantations)/ No. of corpora lutea x 100
Post implantation loss was similarly calculated from the formula:
(No. of implantations - no. of live foetuses)/ No. of implantations x 100
Litter weight and mean foetal weight were calculated from individual foetal weight. Sex ratio was expressed as the percentage of males per litter.

# Group litter values
Group mean values calculated from individual litter values were presented including valid data from all animals with live foetuses at termination (Day 20 of pregnancy).
All derived values (eg percentages, ratios) were first calculated within each litter and the group value derived as a mean of the individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A transient increase in post dose salivation was observed in 4/25 animals treated at 100 mg/kg/day. This sign was noted in each animal on one occasion from Day 11 to 15 post coitum. Increased salivation was not noted at 10 or 50 mg/kg/day. Fur loss was also noted from Day 8 post coitum in 9, 13, 13 and 16/25 animals treated at 0, 10, 50 and 100 mg/kg/day respectively.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight gain showed no effect of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption measurement did not reveal any effect of treatment. The substance was administrated by oral gavage and not in food.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Apart from confirmation of fur loss for 4, 3, 4 and 8/25 animals treated at 0, 10, 50 and 100 mg/kg/day respectively for which it was noted previously, post mortem findings at terminal autopsy were minimal and showed no association with treatment.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
It was considered that there was no effect on litter values. Marginal differences between the controls and animals receiving 100 mg/kg/day seen for implantation rate, embryonic deaths and resulting number of live young were not statistically significant and are considered not to be of toxicological importance, particularly in the absence of a toxic effect to the dam at this dosage level.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
It was considered that there was no effect on litter values. Marginal differences between the controls and animals receiving 100 mg/kg/day seen for implantation rate, embryonic deaths and resulting number of live young were not statistically significant and are considered not to be of toxicological importance, particularly in the absence of a toxic effect to the dam at this dosage level.
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): It was considered that there was no effect on litter values. Marginal differences between the controls and animals receiving 100 mg/kg/day seen for implantation rate, embryonic deaths and resulting number of live young were not statistically significant and are considered not to be of toxicological importance, particularly in the absence of a toxic effect to the dam at this dosage level.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
dead fetuses
effects on pregnancy duration
food consumption and compound intake
mortality
number of abortions
pre and post implantation loss

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number and distribution of malformations, visceral anomalies and skeletal variants, together with the absence of statistically significant differences indicated that there was no treatment-related effect of chlorphenesin on these categories of abnormality.
For skeletal anomalies, the distribution amongst litters was higher at all dosage levels than for controls, attaining statistical significance at 50 mg/kg/day. However, in the absence of a clear dosage-relationship or of an obvious trend in any specific type of skeletal anomaly, these differences were considered to be unrelated to treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number and distribution of malformations, visceral anomalies and skeletal variants, together with the absence of statistically significant differences indicated that there was no treatment-related effect of chlorphenesin on these categories of abnormality.
For skeletal anomalies, the distribution amongst litters was higher at all dosage levels than for controls, attaining statistical significance at 50 mg/kg/day. However, in the absence of a clear dosage-relationship or of an obvious trend in any specific type of skeletal anomaly, these differences were considered to be unrelated to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number and distribution of malformations, visceral anomalies and skeletal variants, together with the absence of statistically significant differences indicated that there was no treatment-related effect of chlorphenesin on these categories of abnormality.
For skeletal anomalies, the distribution amongst litters was higher at all dosage levels than for controls, attaining statistical significance at 50 mg/kg/day. However, in the absence of a clear dosage-relationship or of an obvious trend in any specific type of skeletal anomaly, these differences were considered to be unrelated to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
ca. 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOEL Chlorphenesin (prenatal developmental toxicity in rats) = 100 mg/kg bw/day.

Executive summary:

The test item has been tested to evaluate the effect of on pregnancy and in utero development of the rat. Doses of 0 (Control), 10, 50 and 100 mg/kg were administered to groups of twenty-five time-mated females, by oral gavage once per day, from Days 6 to 15 post coitum inclusive. Clinical signs, bodyweight and food consumption data were collected. On Day 20 all females were killed and subjected to post mortem examination; litter values were determined and foetuses preserved for subsequent examination of visceral and skeletal changes.

The following comments in relation to principal findings during the study are made in summary:

- all dosages were without adverse effect on embryofoetal survival, growth and development in utero;

- there was no evidence of maternal toxicity. Although there was an apparent increase in the incidence of fur loss noted in all treated groups during the live phase of the study, post mortem examination indicated that there was an increase only at the high dosage level, and that the fur loss was mostly classified as minimal in degree and sometimes associated with scabbing. It is therefore considered unlikely that this fur loss was associated with the administration of the test item;

- 100 mg/kg bw/day was associated with transient post dosing salivation for 4/25 females on isolated occasions towards the end of the treatment period;

- maternal bodyweight gain and food intake were not affected at any dosage.

 

In conclusion, treatment of the parent female produced no selective effect on embryofoetal development.

The maximum no observable effect level for selective toxicity to the developing foetus was therefore considered to be 100 mg/kg bw/day. There was no evidence of maternal toxicity.