2-ethylhexyl (4-chloro-2-methylphenoxy)acetate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.08.1999 - 03.06.2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across source

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4-chloro-2-methylphenoxy)acetic acid

EC Number:

202-360-6

EC Name:

(4-chloro-2-methylphenoxy)acetic acid

Cas Number:

94-74-6

Molecular formula:

C9H9ClO3

IUPAC Name:

(4-chloro-2-methylphenoxy)acetic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Experimental animals
Wistar rats (Imp: WIST), males and females, 6-7 weeks old (generation P), were obtained from the Institute of Occupational Medicine in Łódź. It is a conventional breeding. Prior the start of the study the animals were acclimated for 14 days in the test conditions. The animals were individually marked.

Experimental conditions
In the acclimate period and the study period the animals were maintained at 22 ± 2°C and a relative humidity of 50 - 60 %. A 12h light cycle was maintained. The rats were caged in plastic units with metal lid (58 x 37 x 21 cm) bedding with wood plane sterilized by UV. Each cage had a shield with code of the study, dose, start and finish date of the study, sex of the animals and their numbers.

Granulated standard laboratory fodder "Murigran" and drinking water were provided for the rats without limitations. The drinking water was analyzed for chemical impurities and micro-organisms by the supplier.

Administration / exposure

Route of administration:

oral: feed

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

reproductive performance

Remarks on result:

other: In two—generation study on rats it was stated that No-observed-adverse-effect-level (NOAEL) for MCPA is 100 ppm.

Results: F1 generation

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: In two—generation study on rats it was stated that No-observed-adverse-effect-level (NOAEL) for MCPA is 100 ppm.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

not specified

Any other information on results incl. tables

Results

1. Clinical examination

1.1. Clinical symptoms

The parental animals P and F1 in the treated groups didn't differ from the control group in the outer appearance and the behavior. At single parental rats in various groups there was stated bronchial murmur (1 female from group 1 and 1 male from group 2 in generation P; 1 female from group 0 and 1 male from group 3 in generation F1), otitis of internal ear (1 female from group 3 in generation F1), dejection (1 male from group 0, 1 male from group 3 and 1 female from group 3 in generation P) and the decrease in body weight (1 male from group 0 and 1 female from group 2 in generation P).

During the experiment 3 parental animals from generation P died:

2 males from control group and 1 female from group 2. Both males died before mating (after 7 and 8 weeks of experiment) and the female during mating (after 10th week of experiment).

At a couple of young rats in generation F1 exomphalos was stated (1 rat from group 2 and 3 rats in 2 litters from group 3) as well as limbs shortening (2 rats from 1 female in group 1). In group 3 there were observed: dejection and decreased activity (40 rats from 8 litters) and shivering (26 rats from 5 litters).

At young rats in generation F2 dejection and decreased activity were observed (13 rats from 1 litter). In group 3 there were observed: dejection and decreased activity (74 rats from 10 litters), shivering (54 rats from 7 litters) and exomphalos (2 rats from 2 litters).

1.2. Body weight and fodder consumption

In parental generation P, comparing to the control group, statistically important decrease in body weight of males from group 2 (1000 ppm) was stated in 2 and 3 week of the experiment (period before mating). The decrease appeared also in the group 3 (2000 ppm) during entire period before mating. Also in generation F1 in group 3 statistically important decrease in body weight of males was observed during entire period before mating.

In the group of parental females, comparing to the control group, statistically important decrease in body weight of females from group 3 was stated in generations P and F1. The decrease in body weight appeared during entire experiment, that means before mating, during pregnancy and lactation. In generation F1 also statistically important decrease in body weight of parental females from group 2 was observed (during first 2 weeks before mating).

The fodder intake by parental males in generation P before mating was similar as in the control group (with exception of group 3, where during 2 week of experiment there was an increase in fodder intake). In generation F1, comparing to the control group, statistically important decrease in fodder intake was observed in group 3 (in the period before mating) and statistically important increase in fodder intake in group 2 (2 and 9 week of exposure).

At parental females in generation P, before mating, comparing to the control group, statistically important decrease in fodder intake was observed in group 2 in 9 week of exposure and statistically important increase in fodder intake in group 3 in 1, 8 and 9 week of exposure. At generation F1, comparing to the control group, statistically important increase in fodder intake was observed in female group 2 in 2, 5, 9 and 10 week of exposure and statistically important decrease in fodder intake in group 3 during entire period before mating.

The fodder intake by pregnant females in generation P and F1 was similar as in the control group.

During lactation in group 3, comparing to the control females, statistically important decrease in fodder intake was observed from 4th till last day in both P and F1 generation. In group 2 statistically important decrease in fodder intake was observed in last week of lactation (table 8).

2. Pathomornhologic examination

2.1. Macroscopic changes

In the dead rats from generation P (2 from control group and I from group 2) pneumonia punilenta was stated.

In parental animals in generation P liquidated according to the experiment schedule, at single females (1 from control group, 1 from group 2 and 2 from group 3) uterus enlargement was stated. At 1 male from group 2 pneumonia purulenta was stated.

 Experiment schedule, at single females (I from control group, I from group 2 and 2 from group 3) uterus enlargement was stated. At 1 male from group 2 pneumonia purulenta was stated.

By necropsy of parental animals in generation F1, at 1 female from group 3, four resorptions were found in right corner of uterus. Besides, at 14 rats (2 from control group, 3 from group 1, 7 from group 2 and 2 from group 3) hydronephrosis of right kidney was observed. They were 8 males and 6 females.

Young rats from group 3, in both generations F1 and F2 differed from control animals and remaining groups as far as size is concerned (they were significantly smaller). In generation F1, at 2 dead newborns from a female from group 1, front limbs shortening was stated. At offspring liquidated after weaning, in 4 cases (1 from group 1 and 3 from group 3) exomphalos was stated. This change appeared also at 2 rats from group 3 in generation F2. At remaining young rats no pathologic changes were stated.

2.2. Microscopic changes

In the pathomorpliologic examination few no specific changes were stated, as circulatory disturbances, regressive and inflammatory changes. These were observed in the control as well as in the exposed animals, in both generations of parental animals. The circulatory disturbances occured mostly as hyperaemia of liver, kidneys, hypophysis and suprarenal glands and in single cases of ovaries. Also single cases of erythrocytorrhagia and extravasation of blood were stated.

As far as regressive changes are concerned, in kidneys of 2 control rats in generation P glass casts were noticed and, in the same generation, at 1 control female and 1 female from group 1 — atrophy of glomus. At 1 male from group 1 in generation P, partly atrophy of germinal epithelium in the testicles was observed.

 Inflammatory changes (inflammation purulenta) was observed in the uterus of 1 female from group 1 and in the lungs of 1 female and 1 male from group 2 in generation P.

3. Evaluation of reproduction

All the females from generation P (except of 1 female from group 2 that died on the third day of mating) were mated.

In the control group (group 0) three females of 28 weren't pregnant. Among 25 pregnant females, four gave birth to dead pups (there was an extension of delivery period at 1 female). Remaining 21 females brought up the pups till weaning.

In the group 1 two females of 28 weren't pregnant. Among 26 pregnant females, three gave birth to dead pups. Remaining 23 females brought up the pups till weaning.

In the group 2 one female of 28 wasn't pregnant. Among 27 pregnant females, one gave birth to dead pups. 25 of remaining 26 females brought up the pups till weaning.

In the group 3 four females of 28 weren't pregnant. Among 24 pregnant females, three gave birth to dead pups. 19 of remaining 23 females brought up the pups till weaning.

Also all the females from generation F1 were mated according to the schedule. In each group 28 females were mated.

In the control group (group 0) one female of 28 wasn't pregnant. Remaining 27 females brought up the pups till weaning.

In the group 1 two females of 28 weren't pregnant. Remaining 26 females brought up the pups till weaning.

In the group 2 four females of 28 weren't pregnant. Among 24 pregnant females, one gave birth to dead pups. Remaining 23 females brought up the pups till weaning.

In the group 3 three females of 28 weren't pregnant. Among 25 pregnant females, one gave birth to dead pups. 6 of remaining 24 females didn't bring up the pups till weaning, the rest did it.

The indices concerning fertility of parental animals in generations P and F1, that means fertility and pregnancy indices and the length of pregnancy. The fertility and pregnancy indices in exposed groups are similar to the ones in control group in both generations P and F1. Also and the length of pregnancy was similar in exposed groups and in control group in generation P and it varied between 22.0 – 22.4 days. In generation F1, in groups 2 and 3, statistically important extension of delivery period (compared to the control group) was observed. The length of pregnancy varied between 21.6 – 22.3 days.

MCPA in a dose 2000 ppm caused statistically important decrease in number of pups in litter, in both F1 and F2 generations and decrease in number of live born pups in generation F,. No differences were stated between exposed and control groups as far as the number of live born pups in generation F1, the number of dead pups and percentage of males and females in litter are concerned.

The values of the live born index in generations F1 and F2 and the 4-day, survival index in generation F1 are similar in exposed groups and in control group. Statistically important differences, compared to the control group, were stated in group 3 (2000 ppm) in indices: the 4-day survival index in generation F2, the 14-day survival as well as general survival index and lactation index in both generations F1 and F2. Also statistically important difference was stated between generation F1 and F2 concerning the 4-day survival index.

The mortality of pups in generations F1 and F2 till 28 day: the increase in mortality appeared in 3 group in both generations, since the first day. General mortality of young rats in generation F1 amounted to 2.2 % in control group, 2.4 % in group 1, 1.6 % in group 2 and 26.2 % in group 3. General mortality of young rats in generation F2 amounted to 0.3 % in control group, 1.7 % in group 1, 1.2 % in group 2 and 29.2 % in group 3.

The average body weight of pups since birth till weaning: Statistically important decrease in average body weight of the animals (comparing to the control group) since birth till weaning was stated in group 3 in both generations F1 and F2 (with the exception of 14th day of life of generation F2) and in group 2 in the moment of weaning in both generations F1 and F2. On the day of weaning (28^thday of life) the average body weight of pups from group 3 was lower: in generation F1 – 26.9 g and in generation F2 —22.1 g from the average body weight of pups from control group, and in group 2: in generation F1 — 5.3 g and in generation F2 – 5.9g.

3. Evaluation of results

In carried reproduction study even the strongest of used doses of MCPA (2000 ppm) didn't cause lethal effect on parental animals in generations P and F1 that could be connected with application of test substance. Deaths of animals were sporadic and only in generation P. 2 males from control group and 1 female from group 2 died. The reason of the deaths was in every case pneumonia pimiento, the illness typical for laboratory rats.

Few no specific changes noticed during pathomorphologic examination, as circulatory disturbances, regressive, progressive and inflammatory changes were observed in case of the control as well as in the exposed animals.

The limbs shortening appeared only in 2 newborns from I female with the smallest dose in generation P. This change wasn't noticed either at the animals from the groups with higher doses of MCPA or the offspring from generation F2. Thus, the data doesn't indicate that the test substance is the reason of the changes. Also there is no proof that the single cases of umbilical hernia, not connected with the doses and duration of the experiment, are caused by the test substance. Thus, in the conditions of the experiment, MCPA has no teratogenic effect in concentrations 100-2000 ppm. In the other experiments, carried in the Institute of Organic Industry, Branch Pszczyna and concerning teratogenic and embryotoxical effects, also no teratogenic effects were stated at rats whose mothers got MCPA in doses 0.5 – 50 mg/kg b.w. during pregnancy.

The indices concerning fertility of parental animals in generations P and F1, that means fertility and pregnancy, were similar in exposed groups on all levels of intoxication and in control group. This result shows, that MCPA given to rats in fodder in doses to 2000 ppm for two generations doesn't affect negatively reproductive performance of males and females nor does it disturb either the phases of females' sexual cycle or males' spermatogenesis process. In the experiment, statistically significant (compared with control group) extension of pregnancy period was observed in group 2 and 3 in generation F2. Average rat's pregnancy period lasts 22 days (21 – 23 days). The results received in the group 2 – 22.2 days and group 3 – 22.3 days fit in physiological of pregnancy duration.

Statistically important decrease in number of pups per litter in group 3 in both generations F1 and F2 and number of live born in generation F2 indicates harmful effect of MCPA in a dose 2000 ppm on foetus during pre-natal development and its embryotoxical influence in this dose.

The analyse of indices concerning the survival of offspring of parents exposed to MCPA showed the decrease (compared with control group) in 4-day survival index in generation F2, 14-day and general (till 28 day of postnatal life) survival index and lactation index in both generations F1 and F2. Thus, it may be stated that MCPA given to feeding mothers in a dose 2000 ppm is harmful for newborns and that shows in increased mortality. The comparison of mortality of newborns till 28 day shows the existence of correlation with indices concerning survival and it constitutes a proof that MCPA in a dose 2000 given to mothers during the lactation increases mortality of newborns in both generations.

The examination of somatic development of newborns on the basis of increase in body weigh showed that newborns from the group of females with the highest dose (2000 ppm) and newborns of females from the group 2 (1000 ppm) weighed less that control animals on weaning day in both generations F1 and F2. This result shows that MCPA given in doses 1000 and 2000 ppm to females during lactation may delay somatic development of the offspring.

Also the clinic symptoms like dejection, decreased activity and shivering observed in case of offspring of females fed with contaminated fodder in doses 1000 and 2000 ppm constitute the proof of harmful influence of MCPA on growth and development of offspring.

MCPA given in two higher doses, during further growth till maturity (it concerns parental animals P and F1) also reacted toxically (in a dose 1000 ppm — temporarily, in a dose 2000 ppm — during entire period before mating) on growth of young animals what was visible in slower growth (compared with control group), despite almost the same fodder intake of parental animals, exposed and control, in generation P.

Analysing fodder intake in the period before mating, one has to take into account the fact of frequent taking the fodder out of feeder by the animals from groups 2 and 3 and destroying it. It can indicate changed organoleptic characteristics of fodder and this could be a reason for increased fodder intake stated in a couple of cases in these groups of females and males.

Harmful effect of MCPA on body weight increase of the rats was also stated during the other experiments on chronic toxicity and carcinogenicity, carried in the Institute of Organic Industry, Branch Pszczyna .

In the reproduction study, harmful effect of MCPA on pregnant females, observed in group with the highest dose in both generations P and F1 (statistically important lower body weight compared to control group, despite similar fodder intake) is hard to a clear-cut interpretation.

Lower body weight, compared to control group, of females from group 3 during lactation in both generations P and F1 may be connected with lower starting weight and lower fodder intake by the females in this period and lower fodder intake could be, in turn, caused by higher mortality of newborns that influenced females' fodder requirement.

Concluding, on the basis of experiment it can be stated that MCPA in used doses 100 - 2000 ppm didn't influence negatively reproductive performance of males and females, it didn't disturb either the phases of females' sexual cycle or males' spermatogenesis process and didn't cause offspring's developmental defects in pre-natal period. However, in a dose 2000 ppm it seems to have a toxic effect on foetus in pre-natal period, what was indicated by the decrease in number of pups per litter, number of live born pups per litter and lower average body weight of newborns. It also seems to have a toxic influence on the growth and development of offspring, what was indicated by increased mortality of the offspring during the lactation and slower growth in postnatal period.

Applicant's summary and conclusion

Conclusions:

Conclusions:
1.           In the experiment no influence of MCPA on mortality of the parental animals was stated. In a dose 1000 and 2000 ppm MCPA influenced the decrease in average body weight of parental females and males.
2.           In two-generation study with doses to 2000 ppm, MCPA didn't influence negatively reproductive performance of the parental animals and didn't cause developmental defects of offspring F1 and F2,
3.           In a dose 1000 ppm MCPA caused the decrease in average body weight of the offsprings F1 and F2 in the moment of weaning.
4.           In a dose 2000 ppm MCPA caused the decrease in number of pups per litter and average body weight of newborns in generations F1 and F2 and number of live born pups per litter generation F2, what constitutes a proof of its embryotoxic influence.
5.           In a dose 2000 ppm MCPA caused the increase in mortality of the offspring F1 and F2 and had a harmful influence on their growth and development.
6.           In two—generation study on rats it was stated that No-observed-adverse-effect-level (NOAEL) for MCPA is 100 ppm.

Executive summary:

SUMMARY

The two-generation reproduction toxicity study of technical MCPA was conducted in agreement with the OECD Guideline for Testing of Chemicals, Method 416/71. The study had a code: TGR-3/99.

168 Wistar rats were divided in 4 groups, 14 males and 28 females in each. The animals were fed continuously (before mating, during mating, pregnancy and lactation periods, including 2 generations), with the contaminated food containing 0, 100, 1000 and 2000 ppm of technical MCPA. In each generation, parental animals were mated once, inside the group.

In the experiment, technical MCPA didn't influence the mortality of parental animals. In the group 3 (2000 ppm), technical MCPA caused the decrease in average body weight of parental females and males.

In the experiment, technical MCPA didn't influence negatively either fertility functions of the parental animals nor developmental defects of the offspring F1 and F2

In a dose 2000 ppm, test substance caused the decrease in the amount of offspring in the litter and the decrease in average body weight of newborns in both generations F1 and F2.

In a dose 2000 ppm, test substance caused the increase in mortality of newborns in generations F1 and F2 and it influenced negatively their growth and development.

In two—generation study on rats it was stated that No-observed-adverse-effect-level (NOAEL) for MCPA is 100 ppm.