Registration Dossier

Diss Factsheets

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
(1983)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4700 (Reproduction and Fertility Effects)
Version / remarks:
(1985)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US-EPA-FIFRA, Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Guideline 83-4 (1984)
Qualifier:
according to guideline
Guideline:
other: MAFF, 59 NohSan No. 4200, (1985)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Biphenyl-2-ol
EC Number:
201-993-5
EC Name:
Biphenyl-2-ol
Cas Number:
90-43-7
Molecular formula:
C12H10O
IUPAC Name:
2-phenylphenol
Details on test material:
- Name of test material (as cited in study report): Technical grade orthophenylphenol (OPP), Dowicide 1, Preventol O Extra, [1,1'-Biphenyl]-2-ol
- Physical state: light tan and white flakes
- Analytical purity: 99.86% (used through approx. study week 38; Analysis Commission No. 23974); 99.47 % (used after approx. study week 38; Analysis Sample No. 35031DRH81310)
- Impurities (identity and concentrations):
- Composition of test material, percentage of components: Technical grade OPP obtained from Dow Chemical Company and Mobay Corporation was mixed in equal proportions.
- Lot/batch No.: Dowicide 1 (Lots MM850109, MM850110, MM850129, MM850215A, MM850220, and MM850221)/ Preventol O Extra (Samples 7719, 7720, 7721, 7722, and 7723) used through approx. study week 38; Dowicide 1 (Lots MM850110, MM850129, MM850215A, MM850220, and MM850221)/Preventol O Extra (Samples 7955, 7956, 7957, 7958, 7959, and 7960) used after approx. study week 38
- Stability under test conditions: indefinitely
- Storage condition of test material: closed container in a cool dry environment (-23 °C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: (P) 9 wks
- Housing: individually in stainless steel cages suspended over bedding of deotized animal cage board bedding; during gestation females were housed individually in polycarbonate cages with Bed-O-Cobs bedding
- Diet: Purina Rodent Laboratory Chow 5001-4 ETTS form, ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 (October 1985 - August 1986), 18-26 (thereafter)
- Humidity (%): 45-65 (October 1985 - August 1986), 40-70 (thereafter)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: acetone/ corn oil mix
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing procedure: The diet containing the test substance was prepared by dissolving the appropriate amount of test substance for each dose level in acetone and corn oil. The corn oil mixture was then added to the diet, using acetone during diet preparation to rinse the equipment. Corn oil was added at 1% of the diet for all dose groups. To maintain the appropriate dose levels, the concentration of OPP in the diet was adjusted weekly during the premating period based on the group mean body weight and food consumption for each dose group.
During the mating, gestation, and lactation periods, females for each dose group received the concentration of OPP in the diet received by the females during the last week of the premating phase for the respective groups. During the second and third weeks of the F1a lactation phase, females received 1/2 and 1/3, respectively, of the dietary concentration administered during breeding and gestation to avoid overdosing the neonates due to increased maternal food consumption and consumption of chow by the pups. Following the F1a breeding, it was decided that a reduction in the dietary concentration of OPP during the lactation phase was not necessary.
Females had a lower concentration of OPP than the males, therefore, to avoid potential overdosing of females during the mating period males received the same concentration of OPP in the diet as their female cage mates.
Following completion of each breeding period, F0 and F1 males received OPP in the diet based on the mean body weight and food consumption for each dose group during the last week of the respective premating periods.
Until all F1b litters were weaned, weanlings chosen to produce the F2 generations received a diet containing the same concentration of OPP that was given to the F0 rats during the first week of treatment.
- Storage temperature of food: in a freezer at -23 °C
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: F1a breeding: 4 days/week up to 2 weeks; F1b, F2a, F2b breeding: 4 days/week up to 3 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 2 weeks (F1a breeding) or 3 weeks (F1b, F2a, F2b breeding) of unsuccessful pairing replacement of first male by another male with proven fertility for another week.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: in a polycarbonte cage for gestation and lactation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item concentrations in diet were verified by mean of liquid chromatographic analysis. The mean recovery of test item in the diet ranged from 87 to 94% of the nominal dose levels.
Furthermore, the test item in the diet was found to be stable in the samples stored in the freezer since no decline in concentration over time was noticed.
Regarding the distribution of the test item in the diet, the analysis of samples taken from different layers within the mixing bowl revealed that following mixing, the test item was homogeneously distributed within the diet.
Duration of treatment / exposure:
F0 parents: 15 weeks
F1 parents: 10 weeks (including post weaning)
Frequency of treatment:
daily
Details on study schedule:
PRODUCTION OF THE F1 GENERATION
The parental F0 generation consisted of 140 males and 140 females divided into 4 groups of 35 animals/sex each.

PRODUCTION OF THE F2 GENERATION
The parental F1 generation was obtained from a total of 132 F1 male and 134 F1 female pups, which had been randomly selected from the different litters obtained from the F0 dams. Each test group of F1 parental animals consisted of 35 animals/sex, except for the control group, which consisted of 27 male and 29 female F1 animals.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
40, 140, and 490 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
35, 125, and 460 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Parental F0 generation (all test groups including control): 35
Parental F1 generation (all test group without control): 35
Parental F1 generation (control group): 27 males and 29 females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected on the basis of the results obtained from the following studies:
- Hiraga & Fujii (1984). Induction of tumours of the urinary bladder in F344 rats by dietary administration of o-phenylphenol. Fd Chem Toxic 22(11): 865-870
- Hiraga & Fujii (1984). Induction of tumours of the urinary system in F344 rats by dietary administration of sodium o-phenylphenate. fd Cosmet Toxicol 19: 303-310
- Reitz et al. (1983). Molecular mechanisms involved in the toxicity of orthophenylphenol and its sodium salt. Chem Biol Interactions 43: 99-119
- Reitz et al. (1984). Biochemical factors involved in the effects of orthophenylphenol (OPP) and sodium orthophenylphenate (SOPP) on the urinary tract of male F344 rats. Toxicol Appl Pharmacol 73: 345-349

In particular the highest test dose was selected close to the level of 500 mg/kg bw/day that was reported to produce a metabolite considered to be potentially toxic (Reitz et al., 1983 and 1984).

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS
The animals were examined for once daily.
BODY WEIGHT
Body weights of males and females were recorded weekly prior to mating. Body weights of the dams further were recorded during gestation and lactation.
FOOD CONSUMPTION
Food consumption of males and females was recorded weekly prior to mating. Food consumption of the dams further was recorded during gestation and lactation. The mean daily diet consumption in g food/kg bw/d was caclulated.
Oestrous cyclicity (parental animals):
Vaginal smears were taken for 2 weeks from 10 females/dose level prior to mating. The vaginal smears were examined microscopically and classified as diestrous, proestrous, or estrous based on the cytological findings.
Sperm parameters (parental animals):
Sperm parameters were not considered.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
- sex ratio
- number of stillborn pups for each litter (daily)
- number of live pups for each litter (daily)
- weighing of pups at birth
- pup weights during weaning, i.e., on day 4 (before culling; see standardisation of litters), 7, 14 and 21

GROSS EXAMINATION OF DEAD PUPS:
- the lungs of the pups found dead after parturition were examined for their ability to float in water, to distinguish between alive born and stillborn pups.
- culled pups were sacrificed for the purpose of gross pathology.
- F1 weanlings not selected as F1 parents were sacrificed for the purpose of gross pathology.
Postmortem examinations (parental animals):
SACRIFICE
- Adult males (F0 & F1 parents): all surviving animals were sacrificed after completion of weaning of the F1 and F2 generation, respectively.
- Adult females (F0 & F1 parents): all surviving animals were sacrificed after completion of weaning of the F1 and F2 generation, respectively.

GROSS NECROPSY
Following sacrifice, the parental animals were subjected to a complete gross pathological examination.

ORGAN WEIGHTS
The weight of the testis and ovary, and of the liver and the kidneys were recorded.

HISTOPATHOLOGY
The following organs were collected and fixed in 10% neutral buffered formalin for histology: ovaries, testes, vagina, cervix, uterus, epididymides, seminal vesicles, prostate gland, pituitary gland, liver, kidneys, urinary bladder and all gross lesions. Tissue samples to be examined histopathologically were embedded in paraffin, cut into 6 µm thick slices and stained with hematoxylin-eosin.
Postmortem examinations (offspring):
SACRIFICE
- The F1 pups (following culling) and weanlings not selected as parental animals were sacrificed for the purpose of necropsy.
- The F2 offsprings were sacrificed after weaning. It has to be noticed that some F2 females were saved to investigate the heritability of hypotrichosis having appeared within generation F2.

GROSS NECROPSY
Following sacrifice, the animals were subjected to gross pathology.

ORGAN WEIGHTS
Not recorded.

HISTOPATHOLOGY
The following organs were collected and fixed in 10% neutral buffered formalin: ovaries, testes, vagina, cervix, uterus, epididymides, seminal vesicles, prostate gland, pituitary gland, liver, kidneys, urinary bladder and all gross lesions. However only the gross lesions were subjected to histopathological examination. For this purpose, samples were embedded, cut and stained as already described.
Statistics:
The following parameters were subjected to statistical evaluation:
body weights, food consumption, number and percentage of pups born alive, number and percentage of stillborn, pup weight, litter counts, mating and fertility indices, gestation length, viability indices, percentage of male and female pups per litter, organ weights and urinarty bladder measurements.
For statistical evalution, following tests were considered:
Bartlett's test for homogeneity, the analysis of variance (ANOVA), Dunnett's t-test, the Chi-square test and Fischer's Exact test.
Statistical significance was determined at p≤0.05 for all tests.
Reproductive indices:
The mating index and the fertility index were considered.
Offspring viability indices:
The viability index for the pups was determined on day 4, 7, 14 and 21.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
treatment-related mortality: 1 F0 female at 125 mg/kg bw/d
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased body weights in adult F0 and F1 males and females at 460 mg/kg bw/d during pre-mating period
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decreased body weights in adult F0 and F1 males and females at 460 mg/kg bw/d during pre-mating period
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
urinary tract lesions, predominantly in males of F0 and F1 generateion at 460 mg/kg bw/d, incidences were less at 125 mg/kg bw/d and in F0 females at 460 mg/kg bw/d
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: decreased food consumption in adult F0 and F1 males and females at 460 mg/kg bw/d outside pre-mating period

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
Regarding the F0 generation, 6 animals died during the experiment, therefrom 2 females of the control group, 1 male of the low dose (35 mg/kg bw/d) group, 1 female of the mid dose (125 mg/kg bw/d) group and 2 males of the high dose (460 mg/kg bw/d) group. Further, 6 animals needed to be sacrificed in extremis, therefrom 1 female of the control group, 2 females of the low dose group, 2 males of the mid dose group and 1 male of the high dose group. None of these mortalities was considered to be treatment-related, except for the female on the mid dose group; since this female died because of uraemia or kidney failure, a relationship to treatment could not be definitively excluded.
Regarding the F1 generation, 3 animals died during the experiment, therefrom 1 male of the low dose group, 1 female of the mid dose group and 1 female of the high dose group. Further, 4 animals needed to be sacrificed in extremis, therefrom 2 males of the mid dose group and 2 males of the high dose group. None of these mortalities was considered to be treatment-related.

CLINICAL SIGNS OF TOXICITY
No treatment-related clinical signs of toxicity could be evidenced.

BODY WEIGHT
A treatment-related decrease in body weights as compared to controls was reported for the high dose adult males and females at both generation levels, F0 and F1 during the pre-mating period. Body weight gain during gestation and lactation was not affected by the treatment.
During the pre-mating period:
In fact, for the F0 animals of both sexes, body weights were decreased up to 8% below control during the pre-mating period; for the F1 animals, body weights were decreased up to 12 and 11% below control, for males and females, respectively.
For the periods outside pre-mating (incl. gestation and lactation):
In fact, for the F0 animals, body weights were decreased up to 12% and up to 7% below control for females and males, respectively over the remaining periods (outside pre-mating). For the F0 animals, body weights were decreased up to 12% and up to 7% below control for females and males, respectively.

FOOD CONSUMPTION (PARENTAL ANIMALS)
A treatment-related decrease in food consumption (up to 15%) as compared to controls was reported for the high dose adult males and females at both generation levels, F0 and F1 for the periods outside pre-mating. Differences in food consumption during pre-mating were either not consistent or not dose-related and thus, they were not considered to be related to treatment.

REPRODUCTIVE FUNCTION (ESTROUS CYCLE)
No treatment-related effects were noticed.

REPRODUCTIVE PERFORMANCE
No treatment-related effects were noticed for mating index, fertility index and gestation length.

NECROPSY-GROSS PATHOLOGY
External gross lesions such as alopecia, ocular discharge or abnormalities of the teeth were seen in all groups and no relationship to treatment could be evidenced. Treatment-related internal gross lesions were reported regarding the urinary tract at the mid and high dose level (125 and 460 mg/kg bw/d). In fact, renal calculi were reported, which were statistically significantly elevated in the F0 males of the high dose group. Calculi also were found in the urinary bladder of males of the mid and high dose group in the F0 generation, as well as in males of the high dose group in the F1 generation.

NECROPSY-TERMINAL BODY AND ORGAN WEIGHTS
The terminal body weights of males and females from both generation F0 and F1 were statistically significantly reduced at the high dose level (460 mg/kg bw/d) compared to control; this finding was treatment-related. Regarding organ weights, the only finding which was considered related to treatment was an increase in relative kidney weight reported for the males of the F0 and F1 generation, at the high dose level.

NECROPSY-HISTOPATHOLOGY
Histopathology findings confirmed the urinary tract to be a target regarding the toxicity of the test item. In fact, histopathological lesions considered to be treatment-related included a transitional cell hyperplasia seen in the urinary bladder of males (F0 and F1) and females (F0) at the high dose level (460 mg/kg bw/d). Measurements of counting cells/layer were done in order to determine the overall thickness of the transitional epithelial layer. Cell counting revealed a statistically significant increase in the number of cells forming the transitional epithelial layer for the F0 males and females of the high dose level, as well as for the F0 females of the mid dose level (125 mg/kg bw/d). Measurement of the layer revealed a statistically significant increase in the depth of the transitional epithelial layer for the F0 males and females of both the mid and the high dose level (125 and 460 mg/kg bw/d) as well as for the F1 males of the high dose level. At necropsy, a case of urinary bladder papilloma was noticed (1 F0 male of the high dose level), which was considered to be related to treatment. Moreover there was no statistically increased incidence of neoplastic lesions.
Further renal lesions defined as secondary lesions resulting from treatment included transitional cell hyperplasia in the kidney, renal calculi, renal haemorrhage and pyelonephritis, and were reported for the F0 males at the high dose level. These lesions occurred at comparatively higher incidences compared to control. These findings were in accordance with the increase in organ weight as reported for the kidney previously. The incidence of calculi in the urinary bladder also was found to be comparatively increased in the F0 males at the mid- and high dose level, as well as in the F1 males at the high dose level.
It was noticed that compared to the F0 generation which was subjected to treatment starting from 9 weeks of age, the animals of the F1 generation already had been exposed in utero. Nevertheless, the incidence of treatment related renal lesions in the F0 generation was comparatively higher than in the F1 generation. This was explained by the fact that the F0 generation was older than the F1 generation at sacrifice.
Other findings such as ovarian cysts und uterine dilatation were incidental and/or not dose-related and thus not considered to be due to treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
35 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed at the lowest dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects in the urinary tract reported for parental animals: calculi in kidney and/or urinary bladder as well as cell hyperplasia in the urinary bladder at 125 and 460 mg/kg bw/day; increased relative kidney weights in males at 460 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction toxicity
Effect level:
460 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse or treatment-related effects regarding reproduction toxicity were noted.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
There were no treatment-related effects on viability.

CLINICAL SIGNS (OFFSPRING)
No treatment-related abnormalities were noticed.

BODY WEIGHT (OFFSPRING)
The statistically significant decreased body weights correlated with lower body weights observed in the adults and probably reflected consumption of the diet by the pups beginning during the second week of lactation. No effect on body weight development was seen during the first week of lactation. This indicates no effect on early pup development.

SEX RATIO
There were no treatment-related effects on sex ratio.

GROSS PATHOLOGY (OFFSPRING)
Necropsy revealed no treatment-related gross and/or histopathological abnormalities.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Generation:
F1
Effect level:
35 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed at the lowest tested dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Generation:
F1
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects in the urinary tract reported for F1 parental animals: calculi in kidney and/or urinary bladder as well as cell hyperplasia in the urinary bladder at 125 and 460 mg/kg bw/day; increased relative kidney weights in males at 460 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
460 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse or treatment-related effects in the offspring were observed.
Key result
Dose descriptor:
NOAEC
Remarks:
reproduction toxicity
Generation:
F1
Effect level:
460 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse or treatment-related effects regarding reproduction toxicity were noted.

Results: F2 generation

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
460 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed in the offspring.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Regarding systemic toxicity, the results of the present 2-generation study are revealed the urinary tract as a target. The NOAEL regarding systemic toxicity was set at 35 mg/kg bw/d, which was the lowest substance level tested.
Regarding reproduction, none of the examined parameters was altered by the treatment, thus indicating that the test item did not affect reproduction. The NOAEL regarding reproduction was set at 460 mg/kg bw/d, which was the highest substance level tested.
Regarding offspring, treatment-related effects could be evidenced neither in the F1 nor in the F2 generation; thus for offspring the NOAEL also was set at 460 mg/kg bw/d.

The test item had no effect on reproductive performance.

Categories Display