mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 November 2002 to 29 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

EC Number:

240-539-0

EC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Cas Number:

16484-77-8

Molecular formula:

C10H11ClO3

IUPAC Name:

(R)-2-(4-chloro-2-methylphenoxy)propionic acid

Test material form:

solid: particulate/powder

Remarks:

Beige powder

Details on test material:

- Expiry date: 30 April 2014
- Storage conditions: Stored in a sealed container at room temperature (10 to 30 °C) in the dark when not in use.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Glx/BRL/Han)BR

Details on species / strain selection:

The rat was selected as it is a readily available rodent species acceptable to the regulatory authorities and is recommended because of its reproductive characteristics and the quantity of published background data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: At the start of treatment P males and females were approximately 7 to 9 weeks old.
- Weight at study initiation: At the start of treatment P males and females were within the weight range 227.4 to 280.3 g (males) and 166.0 to 210.6 g (females).
- Housing: Animals were housed in groups of up to four (pre-pairing and post-pairing), one female with one male (pairing). During the gestation and post-partum phases, females were housed individually in solid floored polycarbonate cages. The F1 generation animals were housed in groups of five in stainless steel wire mesh cages.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: All animals were given a clinical inspection for ill health on arrival. They were acclimatised for six days and not ten as stated in the protocol. A veterinary inspection was performed before the start of dosing to ensure their suitability for study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70 % relative humidity. On several occasions the humidity was outside the protocol range, the highest recording being 74 %.
- Air changes (per hr): Air-conditioned to provide a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: Test diets of the test material in the vehicle were prepared weekly. The test material was weighed into pre-labelled beakers. The test material was transferred to a mortar containing a small quantity of diet. The test material was then ground down with the diet. The mixture was added directly to the diet in the mixing drum and mixed for 5 minutes.
- Storage temperature of food: The test diets were stored at room temperature (10 to 30 °C) in a sealed container.

Details on mating procedure:

- M/F ratio per cage: One male was housed with one female from the same treatment group.
- Length of cohabitation: After the growth/maturation phase one male was housed with one female from the same treatment group for up to 15 days.
- Proof of pregnancy: Mating was confirmed by the presence of a vaginal plug in situ or sperm in a vaginal washing. On confirmation of mating, vaginal washing was discontinued and the male was removed and rehoused. The day on which mating was confirmed was designated Day 0 of gestation.
- After successful mating each pregnant female was caged: Females were housed individually in solid floored polycarbonate cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity: Stability and homogeneity was performed for each batch of test material used on the study.
- Achieved concentrations: Samples were taken from the dietary formulations prepared for the first and last weeks of treatment of each generation and were analysed for achieved concentration.

Analysis of test diets in Weeks 1, 11, 17 and 22 of treatment showed that they were within the range 89 to 110 % of nominal, with the exception of one replicate sample of the Intermediate dose group formulation in Week 1, where a concentration of 117 % of nominal was obtained. Analysis of a third sample from this formulation gave a value of 101 % of nominal.

Duration of treatment / exposure:

Both sexes were treated for 10 weeks before pairing on a one to one basis within each group. The females continued with this treatment throughout gestation and then were allowed to litter and rear their offspring to weaning. During the lactation period, the females were given diets containing nominal concentrations of 0 (control), 300, 530 and 790 ppm test material. Males were treated until evaluation of the females had been completed. Ten animals of each sex were randomly selected from each group to form the filial (F1) generation. These animals received test material at the original concentrations for 4 weeks post-weaning.

Frequency of treatment:

Continuous in diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm

Remarks:

This dose level was fed during the pre-mating, gestation and post-weaning periods.

Doses / concentrations 2

Dose / conc.:

800 ppm

Remarks:

This dose level was fed during the pre-mating, gestation and post-weaning periods.

Doses / concentrations 3

Dose / conc.:

1 200 ppm

Remarks:

This dose level was fed during the pre-mating, gestation and post-weaning periods.

Doses / concentrations 4

Dose / conc.:

300 ppm

Remarks:

During lactation, the inclusion levels were reduced to approximately 60 % for Group 2 (500 ppm group)

Doses / concentrations 5

Dose / conc.:

530 ppm

Remarks:

During lactation, the inclusion levels were reduced to approximately 66 % for Group 3 (800 ppm group).

Doses / concentrations 6

Dose / conc.:

790 ppm

Remarks:

During lactation, the inclusion levels were reduced to approximately 66 % for Group 4 (1 200 ppm group).

No. of animals per sex per dose:

12 per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Doses were selected by the Sponsor on the basis of previous studies.
In a 90-day rat study, a dose of 1 600 ppm test material in diet reduced growth in both male and female rats with some parallel changes in clinical chemistry (liver and kidney function tests) and haematology (red cell parameters). The target organ was established as the kidney which was increased in weight, both absolute and relative, compared to controls. Changes in clinical chemistry indicated that the liver may also be affected but histopathological change was not seen in either organ. In common with other phenoxy herbicides clinical signs of intoxication are seen only at very high (peri-lethal) doses. Thus the most sensitive index of effect for routine use is bodyweight. At 1 600 ppm the bodyweight effect was slightly greater than that required to establish MTD; a dose of 3 200 ppm was tolerated but effects were clearly in excess of the MTD. Thus, for the current study a high dose of 1 200 ppm was selected. It was anticipated that this would be sufficient to establish an MTD in the parental animals without excessive toxicity to the offspring.
In the 90-day study referred to above there were statistically significant but minor effects on weight gain in female (but not male) rats at a dietary inclusion rate of 800 ppm. The mid dose in the current study was set at this level in order to establish a fall-back position should the high dose prove excessive for the pups and to establish whether there are significant effects on pups at a dose where parental effects are minimal.
The low dose of 500 ppm was the high dose in the previous 2-generation study with the test material. There were only minor effects on the parents. It was anticipated that with the current design there would be a similar slight parental effect (although it may not have been evident with the small group size in this study), but by contrast with the previous study pup effects should be absent.
Compound consumption was calculated using the following formula:

Compound consumption (mg/kg/day) = (diet concentration (ppm) x food intake (g/day)) / mid-interval body weight (g)

- Rationale for animal assignment: The P animals were assigned to treatment groups during the acclimatisation period using a randomisation procedure based on stratified body weight. Allocation of the F1 generation was by random selection of 10 males and 10 females from the available litters.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined twice daily to detect any which were dead or moribund.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined at least once daily for signs of ill health or overt toxicity. Any abnormalities of appearance or behaviour or other signs of reaction to treatment or ill health were recorded and a detailed individual record was maintained of the clinical condition of each animal on the days of body weight recording.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly for the males. For the females, individual body weights were recorded weekly during the growth/maturation phases until confirmation of mating, on Days 0, 7, 14 and 20 of gestation and on Days 1, 4, 7, 14 and 21 post-partum. Individual body weights were recorded weekly during the 4 weeks of maturation of the F1 generation.

FOOD CONSUMPTION AND COMPOUND INTAKE: YES
- The food consumed by each cage of animals was determined weekly during the pre-pairing periods (males and females).
Individual food intake was recorded daily and reported over Days 0 to 6, 7 to 13 and 14 to 19 of gestation (mated females) and over Days 1 to 3, 4 to 6, 7 to 13 and 14 to 20 post-partum (littering females) but reported on the body weight intervals.
Food intake for the F1 generation was measured over the same intervals as the body weights.

WATER CONSUMPTION AND COMPOUND INTAKE: No.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes.
- On Day 4 post-partum, litters were culled to a maximum of eight pups with an equal sex distribution where possible. Animals considered unlikely to survive to weaning were pre-selected for cull and a random selection procedure was used for additional pups.
- Weaning of the F1a offspring: On Day 21, 10 pups/sex were randomly selected from available litters for the F1 generation. Pups not selected were killed and necropsied.
- Maturation phase F1 generation: Selected F1 animals underwent at least 4 weeks of maturation, whilst receiving the control or test material formulations.


GROSS EXAMINATION OF DEAD PUPS:
- All pups found dead were examined macroscopically for structural or pathological changes. Culled pups were examined externally and discarded if normal. Abnormal pups were examined for structural or pathological changes.

Postmortem examinations (parental animals):

- Procedures for post-partum phase: All females were allowed to litter and rear offspring to weaning (Day 21 post-partum). The day pups were first observed was designated Day 0 post-partum. Abnormal signs of nesting, parturition or nursing were recorded.
- All animals were killed by an intraperitoneal injection of sodium pentobarbitone.
- All P animals were examined externally and discarded if found normal. Abnormal animals were examined for structural or pathological changes.
The uterus of all P females was stained in 10 % ammonium sulphide and the number of implantations recorded.

Postmortem examinations (offspring):

All F1 animals were examined externally and discarded if found normal. Abnormal animals were examined for structural or pathological changes.
All pups found dead were examined macroscopically for structural or pathological changes. Culled pups were examined externally and discarded if normal. Abnormal pups were examined for structural or pathological changes.

Statistics:

Body weight gains and food intake were analysed using one-way analysis of variance (ANOVA). Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P ≥ 0.01), pairwise comparisons with control were made using Dunnett's test. A linear contrast was performed to determine whether there was a relationship between increasing dose and response. A significant trend (P < 0.05) was only reported where none of the pairwise comparisons was significant.
The number of implantation sites, number of pups born, percentage of male pups Day 1 and pup weights were analysed using non-parametric methods. The non-parametric methods employed were the Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a dose related trend and the Wilcoxon rank sum test for pairwise comparisons. Where the Kruskal-Wallis ANOVA was not significant, the pairwise comparisons were not reported in order to protect the Type I error.
The proportions of females with post-implantation survival index, live birth index and viability indices of 100 % and gestation, mating, fertility and fecundity indices were analysed using the Cochran-Armitage test for dose-response and Fisher’s exact test for pairwise comparisons. The tests were interpreted with one-sided risk for decreased incidence with increasing dose.

Reproductive indices:

Calculations
Group mean values for certain litter parameters were calculated as:
Mean 1 includes data from all surviving females giving birth including those showing total litter loss post-partum.
Mean 2 includes data from females rearing some young to weaning.

A number of indices were used, where appropriate, to evaluate reproductive function:

Mating index = (number of females with determined copulations / number of oestrous cycles required for their insemination) x 100

Female fecundity index = (number of pregnant females / number of females mated) x 100

Male fecundity index = (number of males siring one or more pregnancies / number of males with one or more confirmed matings) x 100

Female fertility index = (number of pregnant females / number of females paired) x 100

Male fertility index = (number of males siring one or more pregnancies / number of males paired) x 100

Median pre-coital time = time (day) by which half the females in the group had shown evidence of mating

Gestation index = (number of females with live pups / number of pregnant females) x 100

Post implantation survival index = (number of pups born / number of implantation sites) x 100

Live birth index = (number of pups alive Day 1 / number of pups born) x 100

% male pups (sex ratio) = (number of male pups Day 1 / number of pups of determined sex) x 100

Offspring viability indices:

Viability index 1 = (number of pups alive Day 4 before culling / number of pups alive Day 1) x 100

Viability index 2 = (number of pups alive Day 7 / number of pups alive Day 4 after culling) x 100

Viability index 3 = (number of pups alive Day 14 / number of pups alive 7) x 100

Viability index 4 = (number of pups alive Day 21 / number of pups alive 14) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

The nature and intergroup distribution of the clinical observations showed no treatment-related trends.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no deaths in the P generation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain of the high dose males was lower than that of the controls during the first five weeks of treatment and the difference was statistically significant during the pre-pairing period (P < 0.01, Dunnett’s test). Thereafter, growth of the males was essentially similar in all groups.
There was a dose-related reduction in the mean body weight gain of the females during the pre-pairing period and the difference from control was statistically significant in the high dose group (P < 0.01 Dunnett’s test). This overall lower mean body weight gain of the treated females during the pre-pairing period was reflected in lower initial mean body weights on Day 0 of gestation. During gestation, there was a dose-related reduction in mean body weight gain and in the high dose group, the difference from control was statistically significant between Days 0 and 7 (P < 0.001), Days 7 and 14 (P < 0.01) and Days 14 and 20 (P < 0.05, Dunnett’s test).
Again, the lower overall mean body weight gain of the treated groups during gestation resulted in lower mean body weights on Day 1 of lactation, particularly in the high dose group. However, during lactation, body weight gain was generally similar in all groups until the last week when the females in the treated groups gained less weight than the controls and the difference was statistically significant in the high dose group (P < 0.01, Dunnett’s test).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the intermediate and high dose groups, there was a dose-related reduction in male mean food intake of the males throughout the pre-pairing period.
Generally, females in all treated groups ate less than the controls during the pre-pairing period and group mean food intake of the high dose females was markedly lower than control. During gestation and lactation, females in the high dose group ate significantly less than the controls (P < 0.01, gestation, P < 0.05, lactation, Dunnett’s test).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating performance was unaffected by treatment.
The mean duration of gestation was unaffected by treatment. There was a statistically significant, dose-related, reduction in the mean numbers of implantation sites in all treated groups compared to control (P < 0.01, low and intermediate groups; P < 0.001, high dose group, Wilcoxon rank sum test). However, the values for the control, low and intermediate dose groups were higher than expected from the historical control data. Although the mean numbers of pups born were lower in all treated groups compared to control, again, the numbers in the control, low and intermediate dose groups were higher than expected from the background data. Given the small group sizes, the biological significance of the reductions in implantations and pups born in the low and intermediate dose groups is questionable. In the high dose group, the mean number of pups born was significantly lower than control (P < 0.01, Wilcoxon rank sum test). There was no effect of treatment on post-implantation survival index.
One female in each of the low and intermediate dose groups showed total litter deaths but the viability of the offspring in the treated groups was generally similar to that of the controls.
The mean body weight of the treated pups was similar to the controls on Day 1 post partum. Mean body weight gain of the pups in the treatment groups was slightly lower than that of the controls, particularly in the high dose group between Days 7 and 21 post partum, but there was no dose-relationship or statistical significance.
Necropsy examination of the weanling offspring showed only one male pup from the high dose group with slight, unilateral, renal pelvic dilatation.

Effect levels (P0)

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

not determinable

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Although the mean body weights of the F1 males in the high dose group was only slightly lower than that of the controls at the start of this generation, mean body weight gain was significantly lower over the 4 weeks (P < 0.01, Dunnett’s test). Mean body weight gain of the F1 females in the high dose group was only marginally lower than that of the controls and the difference was not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food intake in the high dose group for both F1 males and females was slightly lower than the controls during the 4 weeks of this generation, particularly the females during the last week.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

not determinable

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

59.6 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Compound Consumption

The following mean intakes (mg/kg/day) of test material were achieved in the P generation.

Dose Level (ppm)	500	800	1200
Males pre-pairing	34.5	53.7	82.9
Females pre-pairing	41.0	64.7	98.4
Females gestation	38.2	60.6	88.8
Dose level (ppm)	300	530	790
Females lactation	48.1	85.8	130.2
Mean female	42.4	70.4	105.8
Sexes combined	38.5	62.1	94.4

 

The following mean intakes (mg/kg/day) of test material were achieved in the F1generation:

Dose Level (ppm)	500	800	1200
Males	59.6	98.0	148.4
Females	61.1	101.5	147.7
Sexes combined	60.4	99.8	148.1

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study dietary administration of 1 200 ppm of the test material to adult male and female rats for 10 weeks prior to mating, 790 ppm to the female rats during the lactation period, and again giving 1 200 ppm to the F1 generation for 4 weeks, produced slight adult and pup toxicity in terms of lower body weight gains and food intakes. At these concentrations there were fewer implantation sites compared to control.
At 500 and 800 ppm (reduced to 300 and 530 ppm during lactation), there were marginal effects on adult and pup weight gain and food intake and slightly lower numbers of implantation sites compared to control.
It is considered that reduction of dietary concentration by approximately one third during the lactation period in a 2-generation study, would allow demonstration of a maternal MTD without inducing excessive effects in the offspring.

Executive summary:

The reproductive toxicity of the test material was assessed according to OECD test Guideline 415 and in compliance with GLP.

The objective of the study was to provide a preliminary evaluation of the test material on the reproduction toxicity in the rat, and to define a Maximum Tolerated Dose (MTD) for a possible subsequent 2-generation study. As the compound was known to show a relatively steep dose relationship, dietary inclusion rates were amended during lactation to take account of the increased maternal food consumption at that time.

Groups of 12 male and 12 female rats were given the test material by dietary administration at nominal concentrations of 0 (control), 500, 800 and 1 200 ppm. Both sexes were treated for 10 weeks before pairing on a one to one basis within each group. The females continued with this treatment throughout gestation and then were allowed to litter and rear their offspring to weaning. During the lactation period, the females were given diets containing nominal concentrations of 0 (control), 300, 530 and 790 ppm the test material. Males were treated until evaluation of the females had been completed. Ten animals of each sex were randomly selected from each group to form the filial (F1) generation. These animals received the test material at the original concentrations for 4 weeks post-weaning.

Analysis of samples from the formulations prepared in Weeks 1, 11, 17 and 22 of the study showed that they were suitable for administration to the study animals.

P generation:

All animals survived to the scheduled kill.

There were no clinical observations that were related to treatment. Males in the high dose group gained less weight than the controls during the first 5 weeks of treatment. There was a dose-related reduction in female body weight gain during the pre-pairing period and gestation. Females in the high dose group gained less weight than the controls during the last week of the lactation period. Group mean food intake was lower than control in the intermediate and high dose group males and in the high dose females during the pre-pairing period. During gestation and lactation, females in the high dose group ate significantly less than the controls.

Mating performance and fertility were unaffected by treatment.

Mean duration of gestation was similar in all groups. In the high dose group, there was a reduction in mean number of implantation sites.

Pup viability was unaffected by treatment. Mean pup weight gain was slightly lower in the high dose group compared to control.

F1 generation:

Group mean body weight gain and food intake of the males and females in the high dose group were lower than control over the 4 week treatment period.

 

Under the conditions of the study dietary administration of 1 200 ppm of the test material to adult male and female rats for 10 weeks prior to mating, 790 ppm to the female rats during the lactation period, and again giving 1 200 ppm to the F1 generation for 4 weeks, produced slight adult and pup toxicity in terms of lower body weight gains and food intakes. At these concentrations there were fewer implantation sites compared to control.

At 500 and 800 ppm (reduced to 300 and 530 ppm during lactation), there were marginal effects on adult and pup weight gain and food intake and slightly lower numbers of implantation sites compared to control.

It is considered that reduction of dietary concentration by approximately one third during the lactation period in a 2-generation study, would allow demonstration of a maternal MTD without inducing excessive effects in the offspring.