Propanoic acid, 2-(4-chloro-2-methylphenoxy)-, 2-ethylhexyl ester, (2R)-

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 1989 - 17 April 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

November 1984

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Toxicology Studies pp.45-48, Japan/MAFF

Version / remarks:

1985

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

202-264-4

EC Name:

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

Cas Number:

93-65-2

Molecular formula:

C10H11ClO3

IUPAC Name:

2-(4-chloro-2-methylphenoxy)propanoic acid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: (P) 35±1 wks
- Weight (mean) at study initiation: (P) Males: 126.2 g; Females: 114.7 g; (F1) Males: 100 g; Females: 90 g
- Housing: housed individually in type DK III stainless steel wire mesh cages (during mating periods, the males designated for mating were kept individually in Makrolon cages type M III; the pregnant animals and their litters were also housed in Makrolon type M III cages
- Diet: ad libitum; Kliba maintenance diet rat/mouse/hamster GLP 343 meal
- Water: ad libitum (those animals selected for urinalysis had no access to food and water in the cages used to take urine samples)
- Acclimation periods: 8 days (for P0 animals only)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light):12 hours/12 hours

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: substance preparations were prepared at intervals of not more than 35 days
- Mixing: test substance was mixed with a small amount of food in a Bosch household mixer, an appropriate amount of food was then added to obtain the desired concentration, while mixing was carried out in a laboratory mixer

Details on mating procedure:

- M/F ratio per cage: 1:1 ratio
- Proof of pregnancy: sperm in vaginal smear referred to as "day 0" and the following day "day 1" p.c. (post coitum)
If an animal of the P0 or P1 generation parental animals had not produced any offspring these animals were mated again with control animals to assess their fertility.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content of MCPP-P acid in the test substance/food mixes was determined by HPLC-method.

Duration of treatment / exposure:

whole lifetime (P0 generation), during which two matings and rearings of offspring took place

Frequency of treatment:

fed in diet, continuously

Details on study schedule:

At least 70 days after the beginning of treatment the animals were allowed to mate in a 1:1 ratio (P0). Females were allowed to litter and rear their pups (F1a generation pups) until either day 4, when the pups were culled to 8 pups/litter preferably with 4 males and four females/litter or day 21 after parturition. At least 10 days after the last weaning of the F1a generation pups, the P0 parental animals were mated again in a ratio of 1:1 for the F1b generation and the females were allowed to rear their pups as the F1a generation. After the F1b generation had been weaned, the P0 generation was fasted for 16 hours before sacrifice.
After weaning, the F1a pups were used to establish the F2 generation by choosing 25 males and 25 females from each dose group with all litters being represented if possible. The F1a generation received the test substance at the same concentration as their parents, and at least 98 days after formation of the P1 generation parental animals, the males and females were mated at a ratio of 1:1 avoiding mating of siblings. Females were allowed to litter (F2 pups) and at day 4 after parturition, culling to 8 pups/female was carried out. After the F2 pups had been weaned the P1 generation was fasted for 16 hours before sacrifice.
All pups, which were not used for establishing the next generation, were sacrificed after weaning.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

20 ppm (nominal)

Remarks:

ca. 2 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 ppm (nominal)

Remarks:

ca. 10 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

500 ppm (nominal)

Remarks:

ca. 50 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
On the basis of the results of repeated–dose toxicity studies, which indicated a renal impairment being more pronounced in males than in females in dosages ranging from 450 ppm down to 100 ppm, the following doses were chosen for the reproduction study with MCPP: 20 ppm: as the expected no adverse effect level, 100 ppm: as a concentration with possibly minimal toxic effects (e.g. increased kidney weights), and 500 ppm: as the dose in which toxic effects were expected (e.g. kidney impairment) in the parental animals, but which should not induce mortality in these animals.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
The nesting, littering, and lactation behavior of the dams were generally evaluated in the mornings in connection with the daily clinical inspection of the dams. Only if there were any special findings (e.g., animal could not litter, umbilical cord not cut), these specific findings were documented with the dam concerned.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: once a week
Exceptions:
During each mating period (1st and 2nd matings of the P0 and mating of the P1 generation parental animals) the females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 post coitum. Females without litters were not weighed during the lactation period of the dams used in parallel. After weaning of the last F1a pups the female P0 generation parental animals were weighed weekly until the beginning of the 2nd mating interval (in parallel to the male P0 generation parental animals).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption of the males was not determined any longer after the 10th (P0 generation parental animals) or 14th (P1 generation parental animals) test week until sacrifice. Furthermore, there was no determination of food consumption in the females during the mating period, animals without positive evidence of sperm (during the gestation period of the dams used in parallel) and animals without litter (during the lactation period of the dams used in parallel). Food consumption was not determined between days 14 and 21 after parturition as envisaged in the test guidelines cited under 2.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
On day 4 p.p., the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If it was not possible in single litters to have 4 pups/sex, it was proceeded in such a way that 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Standardization of litters was not performed in litters with ≤ 8 pups.

PARAMETERS EXAMINED
The following parameters were examined in [F1, F2] offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioral abnormalities, anogenital distance (AGD)

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

BEHAVIORAL TESTS (Neurotoxicity)
All surviving pups were tested for the gripping reflex (on day 13), the hearing test (acoustic startle on day 21) and the pupillary reflex test (on day 20).

CLINICAL CHEMISTRY: Yes
Blood was taken from the retro-orbital venous plexus in the morning from non-fasted, not anesthetized animals. The following parameters were determined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, and magnesium

URINALYSIS: Yes
With the exception of the sediment examination and the specific gravity, all the urine constituents were determined semi quantitatively using test strips. The following parameters were examined: Volume, appearance, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, and sediment.

Postmortem examinations (parental animals):

Parental animals of the P0 and P1 generations were subjected to gross pathology after weighing of animals. Organs (liver, kidneys, testes, vagina, cervix, uterus, ovaries, epididymides, seminal vesicles, coagulation gland, prostate, pituitary gland) were all subjected to a histopathological examination in all animals from the control and 500 ppm groups. In addition livers, kidneys, and gross lesions in the 20 and 100 ppm groups were studied likewise.

Postmortem examinations (offspring):

GROSS NECROPSY/HISTOPATHOLOGY / ORGAN WEIGTHS
Culled (i.e. all pups which were sacrificed on day 4 p.p. as a result of standardization) and surplus pups (i.e. all pups reared until day 21 p.p. except those F1a generation pups selected as the basis of the P1 parental generation) were sacrificed on day 21 after birth or subsequent days by means of CO2, examined externally and eviscerated, and their organs were assessed macroscopically. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods. All stillborn pups and all pups that died during rearing were examined externally and eviscerated, and their organs were assessed macroscopically. If there were notable findings or if abnormalities were found in the daily clinical observation of the animals after their delivery, the affected animals were, if it was deemed necessary, examined additionally using appropriate methods.

Statistics:

Dunnett's Test was used for statistical evaluation of all parametric data such as food consumption (parental animals), body weights and body weight change (parental animals and pups).
Fisher's Exact Test was used for statistical evaluation of all quantitative data such as developmental stages, gripping and pupillary reflex, hearing test, number of live and dead pups, and the different indices (e.g. male and female mating index, male and female fertility index, gestation index, live birth index, viability index, lactation index).
Differences between control and treated groups were considered significant at p ≤ 0.05 (indicated with a) or p ≤ 0.01 (indicated with b). For clinical chemistry the means for the dose groups were compared with those for the control group using the analysis of variance (ANOVA and DUNNETT's test). The assessment to whether certain characteristics on urinalyses differed in degree in the control and test groups was carried out using the chi2 test in appropriate two-by-two contingency tables.

Reproductive indices:

Female and male reproduction data were calculated using relevant data and formulas.

Offspring viability indices:

The total number of pups delivered and the number of live born and stillborn pups was noted, and the live birth index was calculated for F1a, F1b and F2 litters.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Only some spontaneous findings without any relation to dose occurred in a few animals of all groups including the controls. Mainly minor skin lesions, swelling of limbs or alopecia were found; furthermore, one control male showed a palpable mass at the flank towards the end of the study and for one 100 ppm male broken incisivi and chromodacryorrhea, which was also noted for one high dose female, were recorded. There were no particular substance-related clinical findings in P0 females during gestation periods for F1a or F1b litters. Without any dose-response relationship insufficient/no nesting activity was observed for several dams of all groups during gestation (F1a and F1b).Moreover, during the gestation period for the F1b pups blood in bedding was found in one high dose female and hemophthalmia in one 100 ppm dam, which died subsequently.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were 2 P0 females (100 ppm) which died spontaneously; these deaths are not associated with treatment. One animal died during gestation of F1b litter (day 10 p.c.) and the other died during the delivery of F1b pups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the whole study the mean body weights/body weight gains of the males and of the females (including gestation and lactation periods) do not show any influence of the test substance on these parameters. All differences between the controls and the animals of the substance-treated groups are assessed to be within the expected range of biological variation. This includes the few isolated statistically significant differences concerning body weight gain between the male controls and the 500 ppm males (week 9-10) and the female control and the 100 ppm group (week 4-5; lactation days 14-21 (F1 a) and week 18-19).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences of biological relevance between the controls and the substance-treated groups concerning the food consumption of the P0 males during the premating period and the food intake of the P0 females during premating, gestation and lactation periods.

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:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The male and female fertility indices for P0 maternal animals are shown in tabes 3 and 4.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Increased mean absolute and relative kidney weights without any correlating morphological findings in next high group (500 ppm)

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

20 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Increased relative mean kidney weight at mid and high dose levels

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive function

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Only some spontaneous findings were recorded in single P1 males and females during premating like microphthalmia (control group), chromodacryorrhea (100 ppm), maloclusion (20 ppm and 500 ppm) and alopecia (500 ppm). No particular clinical findings were noted for P1 dams except no or insufficient nesting activity, which was recorded for several dams of all groups including the control and which occurred without any dose-response relationship.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gains were not adversely affected by the administration of the test substance to the parental animals of test groups (20, 100 and 500 ppm) during the whole study period including gestation and lactation periods of the dams for F2 litters. All differences between the controls and the substance-treated groups are regarded as spontaneous, including the sporadic and sometimes even statistically significantly increased or decreased body weight gains of the females in test groups 20 ppm, 100 ppm and 500 ppm during premating, gestation and lactation, which occurred without any dose-response relationship.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No impairment of food consumption was recorded for the substance-treated P1 parental animals when compared to the controls, neither during the premating period nor during the gestation and lactation period of the P1females. All differences between the groups are without any biological relevance.

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:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In comparison to controls, there was a statistically significant increase in the relative and absolute mean kidney weights of treated animals for which no correlating morphological finding was found at light microscopy (see tables 1 and 2).

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The male and female fertility indices are seen in tables 5 and 6. All pregnant females gave birth to litters with live-born pups. Consequently the gestation index was 100% for all groups. The live birth index was not substantially influenced.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lower relative kidney weights in mid and high dose level

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive function

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: highest dose tested

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No abnormal clinical symptoms were recorded for the F1a and F1b pups. Only some clinical findings (e.g. shortened tail, lesion of hindlimbs and traumatic lesion of cornea) were detected in very few F1a and F1b pups of different groups without a clear dose-response relationship. These findings are finally assessed as spontaneous ones.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of pups, which died or were cannibalized from day 1 to day 4 post partum (before culling), was statistically increased in the 500 ppm group (F1a and F1b) and also marginally increased in the 100 ppm group of F1b in the 100 ppm group in comparison to the control group. As a consequence of the increased pup death from day 1 to 4 post partum in the F1a pups of the 500 ppm group the viability index of this group was significantly reduced. The lactation index, an indicator of pup survival from day 4 to 21 post partum, was not influenced by administration of the test substance, nor was the sex ratio affected (for details see tables 7 and 8).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Only body weight gains of the high dose F1a pups were marginally impaired in comparison to the controls on days 7-14 p.p., which has to be attributed to the test substance administration. The statistically significantly reduced weight gain of the 100 ppm F1b pups, however, is assessed as a spontaneous effect due to the missing dose-response relationship (see table 9).

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of stillborn pups, pups that died intercurrently, culled pups and surplus pups of F1a and F1b litters did not reveal any difference of biological relevance between the test groups either in the type or in the number of pup necropsy observations. The statistically significant increase of 20 ppm pups of the F1b generation, which showed findings at necropsy, is mainly caused by a higher number of pups with dilated renal pelvis; however, this finding is also present in the historical control data. Only very few of the large number of examined pups of all groups showed some other spontaneous findings like incisors sloped, hydroureter and focal liver necrosis.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

As to the several morphological development stages monitored up to weaning there were no biologically relevant differences between the control and the substance-treated F1a or F1b pups. The statistically significantly lower number of F1b pups of test groups receiving 100 and 500 ppm with pinna unfolding on time and the statistically significantly higher number of low dose F1b pups with eye opening on time are not assessed as substance-related effects due to missing dose-response relationship.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1a

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: F1a pubs showed a lower mean pub body weight in comparison to the controls on days 7-14 p.p. at highest dose level

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1b

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slightly increased number of F1b pubs iat mid and high level which died or were cannibalized and reduced viability index

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

For a few pups some spontaneous findings like edema of the hindlimbs, lesion of/or shortened tail and lesion of hind-and/or forelimbs were recorded without any dose-response relationship.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of pups, which died or were cannibalized from days 0-4 p.p. was increased in the 500 ppm-group in comparison to the control group and as a consequence, the viability index as an indicator of the pups' viability during the first 4 days after birth was lowest in this test group; this has to be assessed as a substance-induced effect. The statistically significantly increased number of cannibalized pups in the 20 ppm group, however, is regarded as an incidental finding, which was mainly caused by one dam, which cannibalized already 7 out of the 16 pups cannibalized in total. The lactation index as an indicator how pups were nursed during the rest of their rearing varied for F2 pups between 100% (20 ppm) and 97% (500 ppm) and did not show any substantial differences between the groups (see table 10).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean pup body weights of the 500 ppm group are statistically significantly lower in comparison to the relevant control values on days 14 and 21 p.p.; moreover, weight gain of these pups is impaired on days 4-7, 7-14 and 4-21 p.p., which has to be attributed to the test substance administration.
All other differences between the groups concerning pup body weight data of the F2 generation are of spontaneous nature.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All recorded pup necropsy observations (e.g. incisors sloped, cataract, dilated renal pelvis etc.) occurred without a clear dose-response relationship. They were recorded for a very few pups of different groups with or without involvement of the control group and are assessed as being of spontaneous origin.

Histopathological findings:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The statistically significantly lower number of high dose pups, which showed auditory canal opening on time is probably connected with the retarded, substance-related growth of these pups.
As to the morphological development stages monitored up to weaning, the number of pups of test groups 20, 100 and 500 ppm showing pinna unfolding on time was statistically significantly reduced in comparison to the control group; however, due to missing dose-response relationship and the unexpected high number of control pups with a positive test result, this is finally assessed as an incidental finding. This is also assumed for the lower number of pups of the intermediate dose group with a positive test result on eye opening (see table 12).
In behavioral test, no substantial differences could be noted between the F2 pups of test groups 20,100, 500 ppm and the control pups. The observable differences are without any biological relevance

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F2

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: significant lower mean body weight and higher number of pubs with delayed auditory canal opening at high dose level (500 ppm)

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

The homogeneous distribution of the test substance in the rat food used was also confirmed. According to the results of the concentration control analyses the minimum content of the test substance in the food was generally guaranteed.

Table 1: Mean values of absolute kidney weights (in g) in males and females of the P0 and P1 generation

	0 ppm	20 ppm	100 ppm	500 ppm
Males P0	3.376 ± 0.287	3.38 ± 0.337	3.492 ± 0.281	3.694 ± 0.231**
Females P0	2.098 ± 0.146	2.12 ± 0.186	2.157 ± 0.142	2.286 ± 0.166**
Males P1	3.084 ± 0.239	3.136 ± 0.324	3.326 ± 0.3*	3.562 ± 0.282**
Females P1	2.03 ± 0.16	2.107 ± 0.201	2.048 ± 0.157	2.189 ± 0.181**

*P ≤ 0.05; ** P ≤ 0.01 (Dunnett-Test, two-sided)

Table 2: Mean values of relative kidney weights (in g) in males and females of the P0 and P1 generation

	0 ppm	20 ppm	100 ppm	500 ppm
Males P0	0.61 ± 0.047	0.624 ± 0.042	0.646 ± 0.049*	0.687 ±0.048**
Females P0	0.668 ± 0.043	0.677 ± 0.054	0.685 ± 0.039	0.731 ± 0.051**
Males P1	0.57 ± 0.053	0.6 ± 0.048	0.625 ± 0.052**	0.664 ± 0.043**
Females P1	0.688 ±0.043	0.71 ± 0.048	0.725 ± 0.051*	0.746 ± 0.045**

*P ≤ 0.05; ** P ≤ 0.01 (Dunnett-Test, two-sided)

Table 3: Male fertility indices for P0 males (in %)

	0 ppm	20 ppm	100 ppm	500 ppm
Concerning F1a litter	100	88	96	96
Concerning F1b litter	100	92	96	100

Observable differences concerning the male fertility indices for F1a and F1b between the groups are assessed as being of spontaneous nature and not related to the test substance administration. All values are in the range of the historical control.

Table 4: Female fertility indices and live birth data for P0 females (in %)

	0 ppm	20 ppm	100 ppm	500 ppm
Female fertility index concerning F1a litter	100	88	96	96
Live birth index concerning F1a litter	96	98	97	95
Female fertility index concerning F1b litter	100	96	96	100
Live birth index concerning F1b litter	97	98	96	97

Table 5: Male fertility indices for P1 males (in %)

	0 ppm	20 ppm	100 ppm	500 ppm
Concerning F2 litter	100	100	92	96

Table 6: Female fertility indices and live birth data for P1 females (in %)

	0 ppm	20 ppm	100 ppm	500 ppm
Female fertility index concerning F2 litter	100	100	92	96
Live birth index concerning F2 litter	99	98	97	97

Table 7: Summary of F1a litter data regarding mortality and viability

	0 ppm	20 ppm	100 ppm	500 ppm
Pubs died	6 (1.7%)	2 (0.7%)	9 (2.6%)	9 (2.5%)
Pubs cannibalized	2 (0.6%)	4 (1.4%)	8 (2.3%)	14 (3.9%)b
Pubs dead (day 1 to 4)	7 (2%)	6 (2.1%)	13 (3.8%)	20 (5.9%)a
Pubs surviving days 0 to 4 (viability index)	341 (98%)	277 (98%)	324 (96%)	316 (93%)b
Pubs surviving days 4 to 21 (lactation index)	200 (100%)	169 (100%)	190 (99%)	188 (100%)

Significantly different from control: a= P<0.05; b= P<0.01; pubs dead=pubs died + scarified moribund + cannibalized

Table 8: Summary of F1b litter data regarding mortality and viability

	0 ppm	20 ppm	100 ppm	500 ppm
Pubs died	5 (1.3%)	7 (2%)	13 (3.7%)b	9 (2.3%)
Pubs cannibalized	1 (0.3%)	5 (1.4%)	4 (1.1%)	8 (2%)a
Pubs dead (day 1 to 4)	4 (1%)	7 (2.1%)	9 (2.7%)	11 (2.9%)
Pubs surviving days 0 to 4 (viability index)	379 (99%)	330 (98%)	324 (96%)a	363 (96%)b
Pubs surviving days 4 to 21 (lactation index)	199 (100%)	179 (97%)	172 (98%)	199 (100%)

Significantly different from control: a= P<0.05; b= P<0.01; pubs dead=pubs died + scarified moribund + cannibalized

Table 9: Summary of F1a and F1b pub body weight data in g (days 7-14 p.p.)

	0 ppm	20 ppm	100 ppm	500 ppm
Males (F1a)	18.3 ± 1.42	18.2 ± 1.34	17.9 ± 1.50	17.2 ± 1.15b
Females (F1a)	17.9 ± 1.31	17.7 ± 1.31	17.3 ± 1.42	16.9 ± 1.27a
Males + females (F1a)	18.1 ± 1.34	18.0 ± 1.27	17.6 ± 1.41	17.0 ± 1.19
Males (F1b)	18.2 ± 1.19	17.8 ± 1.36	16.9 ± 2.27b	17.3 ± 1.75
Females (F1b)	17.7 ± 1.12	17.3 ± 1.36	16.1 ± 2.61b	17.0 ± 1.72
Males + females (F1b)	18.0 ± 1.12	17.5 ± 1.31	16.5 ± 2.34a	17.2 ± 1.71

Significantly different from control: a= P<0.05; b= P<0.01

Table 10: Summary of F2 litter data regarding mortality and viability

	0 ppm	20 ppm	100 ppm	500 ppm
Pubs died	14 (4.3%)	6 (2.1%)	21 (6.9%)b	28 (7.8%)
Pubs cannibalized	3 (0.9%)	16 (5.5%)b	5 (1.6%)	10 (2.8%)
Pubs dead (day 1 to 4)	16 (5%)	20 (7%)	22 (7.4%)	19 (5.5%)
Pubs surviving days 0 to 4 (viability index)	305 (95%)	262 (92%)	275 (93%)	314 (91%)
Pubs surviving days 4 to 21 (lactation index)	193 (99%)	179 (100%)	167 (98%)	183 (97%)

Significantly different from control: a= P<0.05; b= P<0.01; pubs dead=pubs died + scarified moribund + cannibalized

Table 11: Summary of F2 pub body weight data in g (days 4-21 p.p.)

	0 ppm	20 ppm	100 ppm	500 ppm
Males (F2)	46.8 ± 4.07	45.1 ± 3.53	45.2 ± 3.25	43.4 ± 5.17a
Females (F2)	43.8 ± 3.50	42.5 ± 3.22	42.5 ± 3.33	40.5 ± 5.90a
Males + females (F2)	45.2 ± 3.64	43.8 ± 3.26	43.8 ± 3.18	41.9 ± 5.59a

Significantly different from control: a= P<0.05; b= P<0.01

Table 12: Summary of pubs physical development (F2 litter)

	0 ppm	20 ppm	100 ppm	500 ppm
Pinna unfolding: Litters tested Pubs tested Pubs reaching criteria
	25	25	22	24
	305	262	275	314
	302 (99%)	236 (90%)b	254 (92%)b	290 (92%)b
Auditory canal opening Litters tested Pubs tested Pubs reaching criteria
	25	25	22	24
	193	179	167	183
	191 (99%)	175 (98%)	167 (100%)	171 (93%)b
Eye opening
Litters tested	25	25	22	24
Pubs tested	193	179	167	183
Pubs reaching criteria	185 (96%)	169 (94%)	149 (89%)a	171 (93%)

Significantly different from control: a= P<0.05; b= P<0.01

Applicant's summary and conclusion

Conclusions:

The continuous administration of MCPP-P acid to rats as a constant homogeneous addition to the diet over two generations caused only marginal signs of systemic toxicity in the highest dose group (500 ppm) in F0 and F1 parents (increased absolute and relative kidney weights without any correlating morphological findings).
Adverse substance-induced effects which were noted for the progeny of the F0 and/or F1 parents were an increased pup mortality in the 500 ppm (F1a, F1b and F2 litters) and 100 ppm (F1b litters only) groups and retarded growth/development of the high dose F1a and F2 pups.
20 ppm were tolerated by both parental generations and their offspring without any changes which could be causally related to the test substance administered.
MCPP-P acid had no adverse effects on reproductive parameters or reproductive organs of the parental animals of both generations (F0 and F1) and of all groups (20, 100 and 500 ppm).
The NOAEL concerning reproductive function is 500 ppm (corresponding to 50 mg/kg bw/day).
The NOAEL concerning systemic toxicity for F0 and F1 parental animals as well as for developmental toxicity for the F1 and F2 progeny is 20 ppm (corresponding to 2 mg/kg bw/day). Thus, there was no indication for specific developmental toxicity as the effects in the progeny occurred at a dose level of parental toxicity.

Executive summary:

A two-generation reproduction toxicity study according to OECD TG 416 and GLP was conducted with MCPP-P acid (CAS 7085-19-0, racemic form). Groups of 25 male and female Wistar rats were given 0, 20, 100, or 500 ppm (0, 2, 10 and 50 mg/kg bw/day) test substance in the diet for their whole lifetime (P0 generation). At least 70 days after the beginning of treatment, P0 animals were mated to produce a first litter (F1a) and subsequently re-mated to produce a second litter (F1b, retained only until weaning). Groups of 25 males and 25 females selected from F1a pups as P1 parental generation were offered diets containing 0; 20; 100 and 500 ppm of the test substance post weaning, and the breeding program was repeated to produce F2 litter. For all parental animals, food consumption was recorded and animals were assessed by gross pathology, histopathological examination, analysis of blood and urinalysis. In the highest dose group of 500 ppm, both parental generations showed increased absolute and relative kidney weights in both sexes. In the mid-dose group, increased relative kidney weights were detected for males only in the P0 animals and increased absolute (males only) and relative (both sexes) kidney weights in the P1 parental animals. In the lowest dose group of 20 ppm, both parental generations did not show any abnormalities concerning clinical and clinicochemical examinations, urinalysis and pathology. The NOAEL for systemic maternal toxicity is 100 ppm for P0 females and 20 ppm for P0 males and 20 ppm for both sexes of the P1 parental generation. For the F1a and F1b pubs, a slightly increased number of pubs which died or were cannibalized (days 0-4 p.p.) and consequently reduced viability indices were observed at the highest does group of 500 ppm accompanied by marginal impairment of F1a pub body weight gain between days 7-14 p.p. For the F2 pubs at this dose level this was observed as well. In addition, F2 pubs showed a lower mean pub body weight in comparison to the controls on days 4-7, 7-14 and 4-21 p.p. and higher number of pubs with delayed auditory canal opening. At 100 ppm, only F1 pubs showed slightly increased number of F1b pubs which died or were cannibalized and reduced viability index, F2 pubs showed nothing abnormal. For the lowest dose group, F1 and F2 pubs showed no abnormality.

Finally, the continuous administration of MCPP-P acid to rats as a constant homogeneous addition to the diet over two generations caused only marginal signs of systemic toxicity in the highest dose group (500 ppm) in F0 and F1 parents (increased absolute and relative kidney weights without any correlating morphological findings).

Adverse substance-induced effects which were noted for the progeny of the F0 and/or F1 parents were an increased pup mortality in the 500 ppm (F1a, F1b and F2 litters) and 100 ppm (F1b litters only) groups and retarded growth/development of the high dose F1a and F2 pups.

20 ppm were tolerated by both parental generations and their offspring without any changes which could be causally related to the test substance administered.

MCPP-P acid had no adverse effects on reproductive parameters or reproductive organs of the parental animals of both generations (F0 and F1) and of all groups (20, 100 and 500 ppm).

The NOAEL concerning reproductive function is 500 ppm (corresponding to 50 mg/kg bw/day).

The NOAEL concerning systemic toxicity for F0 and F1 parental animals as well as for developmental toxicity for the F1 and F2 progeny is 20 ppm (corresponding to 2 mg/kg bw/day). Thus, there was no indication for specific developmental toxicity as the effects in the progeny occurred at a dose level of parental toxicity.