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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 1987 - February 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
30 male and 30 female Sprague-Dawley rats per group and generation (P and F1) were treated with zinc naphthenate. The dosage groups were as follows: 0, 500, 1000 and 5000 ppm in the diet. The vehicle was corn oil. In addition, a vehicle control group was also tested. The animals were treated 10 weeks prior to mating (males/females), during mating (males/females), gestation (females) and lactation (females).
GLP compliance:
yes
Remarks:
US-GLP (Title 40, Code of Federal Regulations, 1985 rev., Part 160, Good Laboratory Practice Standards
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: semi-solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Sprague Dawley COBS, CD rats
- Source: Charles River Laboratories of Wilmington, Massachusetts.
- Age at study initiation: 7 weeks of age
- Housing: animals were kept three per cage,
- Diet (ad libitum): certified rodent ration (Zeigler Bros., Inc., Gardners, Pennsylvania). The ration arrived in the form of lab block and was ground to a uniform consistency.
- Water (ad libitum): water
- Quarantine period: a 2-week quarantine; Body weights and feed consumption were monitored during this pretreatment period.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1°C (18.3 - 23.9°C)
- Relative humidity: 50% (40% - 60%)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Remarks:
Mazola
Details on exposure:
DIET PREPARATION
Feed was prepared on a weekly basis by dissolving the appropriate amount of zinc naphthenate in corn oil with the aid of heat, while keeping the total volume of corn oil/zinc naphthenate for each dosage group constant. These solutions were, in turn, poured into ground rodent ration and thoroughly blended with a mechanical mixer. To ensure homogeneity and accuracy of the preparation, samples were extracted from each batch of feed and analysed for zinc content by atomic absorption.

Compound concentration in feed remained at a constant level throughout the entire study with no attempts to adjust for body weight gains.
Details on mating procedure:
- M/F ratio per cage: a female with a male of the same dosage group.
- Proof of pregnancy: mating success was checked daily and was determined by the presence of sperm plugs on cage pads.
- After successful mating each pregnant female was caged (how): when a positive mating was achieved, the females were removed from their wire cages and housed individually in polycarbonate boxes, where bedding material was provided.
- Animals not showing evidence of mating after 1 week were paired with a male of the same dosage group that had successfully mated.
- Further matings after two unsuccessful attempts: yes, after the second week, females failing to mate were given mates that were proven. Following the third week of mating, all remaining females for which there was no evidence of mating were also placed in polycarbonate boxes.

Mating of the F1 rats was accomplished in the same manner as the P rats, with the exception of the care taken to avoid mating rats of the same litters.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To ensure homogeneity and accuracy of the preparation, samples were extracted from each batch of feed and analysed for zinc content by atomic absorption.
Compound concentration in feed remained at a constant level throughout the entire study with no attempts to adjust for body weight gains.
Duration of treatment / exposure:
1) P generation:
Males:
- 10 weeks prior to mating
- throughout mating until necropsy
Females:
- 10 weeks prior to mating
- throughout mating, gestation and lactation

2) F1 generation:
Males:
- 10 weeks prior to mating starting after weaning
- throughout mating until necropsy
Females:
- 10 weeks prior to mating
- throughout mating, gestation and lactation
Frequency of treatment:
ad libitum
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500ppm (= 25mg/kg bw/day)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1000ppm(= 50mg/kg bw/day)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5000ppm(= 250mg/kg bw/day)
Basis:
nominal in diet
No. of animals per sex per dose:
P generation: 30 males / 30 females
F1 generation: 30 males / 30 females
Control animals:
yes, concurrent vehicle
Details on study design:
PILOT STUDY
The dosages of zinc naphthenate in the main study were based on the previously conducted pilot study which indicated effects between 2,700 ppm and 5,000 ppm in both parental sexes and their offspring.
The pilot study is described as follows:
A pilot study was conducted using 72 Sprague Dawley COBS, CD rats (six groups, each of six male and six female). Dosages for the study were calculated on a percent diet basis and were derived from expected food consumption and toxic signs seen in the acute oral studies. The concentration of zinc naphthenate in the feed ranged from 0.13 percent (1,300 ppm) in the low dosage group to 2.10 percent (21,000 ppm) in the high dosage group.
Results:
Male and female rats receiving 1 and 2 percent zinc naphthenate in their diet (10,000 ppm and 20,000 ppm) showed reduced body weights following 4 weeks of compound administration. Both sexes of rats in these groups also became lethargic and experienced urogenital staining from compound
consumption. Mating of rats within their respective groups indicated the 2 percent zinc naphthenate group required twice the mating time as did the other five dosage groups. Litters born of the highest two dosage groups (one and two percent) experienced reduced birth numbers and reduced total birth weights. At the time of weaning, offspring numbers and weights for these high dosage group litters were greatly reduced when compared to
litters of lower dosage groups and controls.
Positive control:
not stated

Examinations

Parental animals: Observations and examinations:
1) P GENERATION

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were checked daily for toxic signs during the 10 weeks before mating.
Dams were checked daily for new births or birthing complications.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded three times per week during the 10 weeks before mating.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

2) F1 GENERATION
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during the 10 week exposure period prior to mating.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: three times a week during the 10 week exposure period prior to mating

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
F1 GENERATION
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes; litters were standardised by randomly culling to four males and four females each, or as close to equal numbers of sexes as permitted. Litters with less than eight pups on day 4 remained intact.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Individual body weights, abnormalities, mortalities, and total litter weights were noted on day 0, 4, 7, 14, and 21 post partum.

Postmortem examinations (parental animals):
1) P GENERATION
SACRIFICE
- Male animals: animals were euthanized by CO2, examined grossly, and tissues removed (testes, epididymides, seminal vesicles, prostate, pituitary, liver, kidneys), for histopathologic examination
- Maternal animals: the dams were sacrificed by CO2 at the time of weaning of the F1 generation. All dams were examined grossly while the vagina, uterus, ovaries, pituitary gland, liver, and kidneys were removed for histopathologic examination.

2) F1 GENERATION
- Male animals: following the 3-week mating period, F1 males were necropsied, taking sex and target organs for histopathologic examination.
- Maternal animals: F1 dams had organs taken for histopathologic examination.

In addition to the periodic sacrifices mentioned above, any animal dying spontaneously or for some reason removed from the study at an early date was submitted for gross necropsy. Gross necropsies consisted of the examination of all external surfaces, orifices, brain and spinal cord, thoracic, abdominal and pelvic cavities, and organs therein.

Postmortem examinations (offspring):
1) F1 GENERATION
SACRIFICE
- The F1 pups not used for further mating were submitted for gross only necropsy at weaning.

2) F2 GENERATION
SACRIFIC
- At weaning, five F2 males and five F2 female pups were selected from each group for removal of sex organs, liver, and kidneys for histopathology. The remaining F2 pups were examined grossly.

In addition to the periodic sacrifices mentioned above, any animal dying spontaneously or for some reason removed from the study at an early date was submitted for gross necropsy. Gross necropsies consisted of the examination of all external surfaces, orifices, brain and spinal cord, thoracic, abdominal and pelvic cavities, and organs therein.
Statistics:
Student "t" probability test (comparison of litter size)
Reproductive indices:
Mating days
Gestation duration
Fertility index
Gestation index
Offspring viability indices:
Viability index
Lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

1) P GENERATION

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Male rats in the 0.5 percent dosage group appeared to become lethargic after week 9 of the dosing period. Alopecia was prevalent in the mid- and high-dosage animals of both sexes.
One male rat from the 0.1 percent dosage group was found cannibalised while the rats were group housed, prior to mating. Fighting was the apparent cause of death.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The P generation female rats in the 0.5 percent group (5000 ppm) experienced significantly reduced body weights by week 3 of compound administration, and continued through week 10. The control, low, and mid-dosage groups gained at similar rates.
Male rats fed a diet of 5000 ppm zinc naphthenate demonstrated significantly decreased body weights by week 8 of the 10 week dosing period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Weekly monitoring of feed consumption during the 10 week exposure period indicated all female rats ate similar portions, regardless of the dosage group. The same was also true for male rats, regardless of the zinc naphthenate concentration in the feed.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The fertility index for all dosage groups was comparable, with pregnancies resulting in between 79 and 86 percent of all rats. All dams produced live litters, with pups in all dosage groups remaining viable through lactation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination of P generation rat tissues revealed compound-related lesions in the kidneys of male rats of the 0.5 percent dosage group. These lesions consisted of accumulations of amorphous to slightly granular, lightly eosinophilic material in the lumina of renal tubules, particularly near the corticomedullary junction. The term "nephrosis" was used to describe the lesions which were considered microscopically distinct from the intraluminal accumulations of homogenous proteinic material associated with the spontaneous degenerative nephropathy syndrome of rats. "Epithelial regeneration" of the renal tubules was evident, which may be assoicated with a variety of degenerative processes, especially the spontaneous degenerative nephropathy syndrome, mentioned above. However the increased incidence of this regeneration observed in the high-dosage rats suggests a compound-related effect. these lesions were not so advanced as to expect a clinically detectable effect on renal function. Lower concentrations of compound did not produce these effects, and all other findings were considered incidental.

2) F1 GENERATION
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One dam, a control group animal, was found dead at approximately 17 days of pregnancy, and was submitted for necropsy. No apparent reason could be determined for the cause of death.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights of female rats in the 0.5 percent dosage group were significantly depressed from the first week of the dosing period through week 10, when compared to the control group weights. Elevated body weights were observed in the 0.05 percent group from weeks 5 through 10.
As with the female rats, male rats in the 0.5 percent dosage group experienced significantly reduced lowered body weights throughout the pre-mating period. Again, male rats in the 0.05 percent group exceeded the body weights of the control and mid-dosage groups during weeks 7 through 10.

FOOD CONSUMPTION
Feed consumption figures during the dosing period revealed that all groups of rats, both male and female, ate similar portions of chow, and were not influenced by compound concentrations.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating time for all F1 rats proved to be of a longer duration than the P generation, with an average of 4.8 days before signs of mating were observed. Mating was attempted with 29 - 30 rats in each dosage group. Pregnancies resulted in 67 percent and 60 percent of the control and 0.05 percent groups, respectively, while 93 percent and 97 percent of the 0.1 percent and 0.5 percent animals became pregnant. All pregnancies resulted in live litters with the exception of one 0.5 percent dam, which produced all stillborn foetuses after being unable to deliver at the predicted time.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination of tissues form the F1 generation rats again showed "nephrosis " and " tubular regeneration" in the kidneys of a small number of male rats which was apparently associated with compound administration. All other findings were considered incidental or associated with spontaneous disease complexes of rats.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for general toxicity based on effects on body weights.
Dose descriptor:
NOAEL
Effect level:
> 5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for reproduction

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

F1 GENERATION
VIABILITY (OFFSPRING)
One dam of the 0.5 percent dosage group delivered all dead pups as a result of her water bottle not being filled over the weekend period prior to her delivery. As a result, the dam was removed from the study and was not included in any statistical determinations.

CLINICAL SIGNS (OFFSPRING)
Alopecia was prevalent in the mid- and high-dosage animals.

BODY WEIGHT (OFFSPRING)
A comparison of litter size showed a significant reduction in the high-dosage group when compared with that of the control group. Due to the fewer numbers of pups born to the 0.5 percent group dams, litter birth weights were also decreased to a significant degree. However, individual pup weights in this high-dosage group were similar to those of the control group, while the pup weights of the low-dosage group were found to be slightly higher.
At weaning, pups of the 0.5 percent dosage group had significantly lower body weights than the control, 0.05 percent or 0.1 percent dosage groups.

2) F2 GENERATION
VIABILITY (OFFSPRING)
Pup survivability was similar for all dosage groups, through lactation.

BODY WEIGHT (OFFSPRING)
Litter size and weights at birth were alike for all dosage groups. At weaning, individual pup weights as well as litter weights were significantly lowered in the 0.5 percent group.

GROSS PATHOLOGY (OFFSPRING)
One control group dam gave birth to a pup suffering from craniorachischisis partialisis, which was considered to be spontaneous abnormality, not associated with the test regimen.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for general toxicity based on effects on body weights.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for reproduction

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL general toxicity: 1000 ppm (corresponding to 50mg/kw bw/day) based on effects on body weight.
NOAEL reproduction: > 5000 ppm(corresponding to 250mg/kw bw/day)
P generation male and female rats fed with a high dose diet containing 5000 ppm zinc naphthenate showed body weight depression even though food consumption was comparable to other groups.
Lethargy was observed in males of the 5000 ppm group, but did not affect their breeding performance. Fertility indices of all groups of P generation animals showed similar fertility indices.
Pups of the 5000 ppm group were of normal size at birth, but showed a significantly lowered body weight, due to maternal stress by the time of weaning. The reduced litter size at birth observed in the high dose group may also be attributed to maternal stress.
Effects on mating success in the control and low dose groups, but not in the mid and high dose group F1 generation animals were not attributed to treatment with zinc naphthenate
In conclusion, continuous feeding of male and female rats with up to 5000 ppm in the diet over two generations did not adversely affect the reproductive function and outcome.