1-Octadecanaminium,N,N-Dimethyl-N-Octadecyl-,(Sp-4-2)-[29h,31h-Phthalocyanine2-Sulfonato(3-)-N 29,N 30,N 31,N 32]Cuprate(1-)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between October 11th, 2004 and October 30th, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to guideline.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 416, ECB Annex V, B35

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: Blue powder
- Analytical purity: 86.8%
- Impurities (identity and concentrations):
Free quaternary ammonium Cmpds.: 2.9%
Copper Phthalocyanine blue: 9%
Water: 1.3%
- Purity test date: 2004-12-30
- Lot/batch No.: F94555
- Expiration date of the lot/batch: 2009-09-22

Test animals

Species:

rat

Strain:

other: Crl:WI(Glx/BRL/Han)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 6-8 Weeks
- Weight at study initiation: At the start of treatment P males and females were approximately 6 to 8 weeks old and were within the weight range 164.3 to 222.8 g (males) and 123.6 to 166.5 g (females). The F1 animals were 29 to 33 days old and within the weight range 60.3 to 118.9 (males) and 57.5 to 107.9 (females) at the start of treatment.
- Fasting period before study: None
- Housing: Animals were housed in groups of up to four (pre-pairing and post-pairing), one female with one male (pairing) or individually (females in the gestation and post-partum phases) in cages that conform to the ‘Code of practice for the housing and care of animals used in scientific procedures’ (Home Office, London, 1989).
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period:14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%):40 to 70%
The temperature recorded in the experimental room was below the protocol range on one occasion (minimum 17.2C) and the relative humidity fell below the specified range on two occasions (minimum 35%). These minor deviations were considered not to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12 hours light (0600 to 1800) and 12 hours dark.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: SQC Rat and Mouse Breeder Diet No 3, Ground Fine (Special Diet Services Ltd. Witham).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): SQC Rat and Mouse Breeder Diet No 3, Ground Fine
- Storage temperature of food: The test diets were stored at room temperature (10 to 30°C) in a sealed container.

VEHICLE
- Justification for use and choice of vehicle (if other than water): SQC Rat and Mouse Breeder Diet No 3, Ground Fine (Special Diet Services Ltd. Witham).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF FORMULATIONS
Formulation analysis
Formulations were analysed by Butterworth Laboratories Ltd. for their test article content by atomic absorption spectrophotometry, using a method (number 14-034A, revised 21 September 2004) provided by the Sponsor.
Stability and homogeneity
Stability and homogeneity analysis were performed for each batch of test article used on the study. As soon as possible after preparation, three samples were removed (one from the top, middle and bottom of each formulation) and stored frozen (-20C) pending analysis. After the initial sampling formulations were allowed to stand at room temperature in the Formulations Analysis laboratory protected from light. After 24 hours and one week, random samples were removed from each formulation and stored frozen (-20C) pending analysis to determine stability.
Achieved concentrations
Samples were taken from the dietary formulations prepared for Weeks 1 and 10 of each of the P and F1 generations. The normal target range for the results was 85% to 110% of nominal concentration. Three samples were removed from each of the test formulations, frozen immediately (-20C) and sent for analysis. An additional sample was removed from each test formulation, frozen immediately (-20C) and stored until further notice.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 15 days
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: None.

Duration of treatment / exposure:

10 weeks prior to mating, during the pairing period, and until the day prior to necropsy

Frequency of treatment:

Daily

Duration of test:

- F1 parental animals not mated until [10] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [21] days of age.
- Age at mating of the mated animals in the study: [14] weeks

No. of animals per sex per dose:

24

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were set based on a 28-day toxicity study in the rat, where a dose level of 1000 mg/kg/day was shown to
increase relative spleen and liver weights and cause slight fore-stomach
hyperkeratosis, a slight increase in spleen haematopoiesis in males and a slight increase in the severity of small foci of hepatic necrosis in males. The NOEL in that study was 200 mg/kg/day.
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No.
- Time schedule for examinations:

OTHER: Clinical pathology

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other:

Fetal examinations:

- External examinations: No
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Statistics:

All variables were analyzed w/ a two-sided risk except:
P & F1: Body weight (BW) gain, food consumption, necropsy BW, hematology, clinical chemistry & seminology were analyzed by ANOVA. Levene's test was used for equality of variances among the groups. Where this showed no heterogeneity (P≥0.01), pairwise comparisons w/ control were made using Dunnett's test. A linear contrast was used to determine dose/response relationship. Where data was available for only 2 groups, a 2 sample t-test was used. The # of implantation sites & pups born, % of male pups Day 1, pup weights & phys. dev. data (F1 only) were analyzed by non-parametric methods (Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a dose related trend, & the Wilcoxon rank sum test for pairwise comparisons). Non-parametric methods were also used in place of the 1-way ANOVA for clinical chemistry parameters with values > or < the limit of the assay. Gestation, fertility, fecundity & mating indices & the proportion of females with post-implantation survival index, live birth index & viability indices of 100% were analyzed by the Cochran-Armitage test for dose-response & Fisher’s exact test for pairwise comparisons. The tests were interpreted w/ 1-sided risk for decreased incidence w/ increasing dose. Organ weights were analyzed by ANCOVA & Dunnett's test, for each sex separately, using the necropsy BW as covariate. Levene's test for equality of variances was also performed. Where this showed evidence of heterogeneity (P generation uterus weights; P<0.01) the organ was analyzed using 1-way ANOVA on absolute organ weights & organ to necropsy BW ratios. F1: FOB & locomotor activity data were analyzed by a 2-sample t-test. Levene's test for equality of variances between the groups was performed. Swimming maze data (escape times) were analyzed using the Wilcoxon rank sum test. A value of >120 was treated as 120 for statistical analysis. The proportion of error-free escapes was analyzed using Fisher's exact test.

Indices:

Mating index = (number of females with determined copulations ÷ number of estrous cycles required for their insemination) x 100
Female fecundity index = (number of pregnant females ÷ number of females mated) x 100
Male fecundity index = (number of males siring one or more pregnancies ÷ number of males with one or more confirmed matings) x 100
Female fertility index = (number of pregnant females÷ number of females paired)x 100
Male fertility index = (number of males siring one or more pregnancies÷ number of males paired)
x 100
Median pre coital time = time (day) by which half the females in the group had shown evidence of mating
Gestation index = (number of females with live pups÷ number of pregnant females) x 100
Post implantation survival index = (number of pups born ÷ number of implantation sites) x 100
Percent male pups (sex ratio) = (number of male pups Day 1 ÷ number of pups of determined sex) x 100
Live birth index = (number of pups alive Day 1÷ number of pups born) x 100
Viability index 1 = (number of pups alive Day 4 before culling ÷ number of pups alive Day 1) x 100
Viability index 2 = (number of pups alive Day 7 ÷ number of pups alive Day 4 after culling) x 100
Viability index 3 = (number of pups alive Day 14 ÷ number of pups alive Day 7) x 100
Viability index 4 = (number of pups alive Day 21 ÷ number of pups alive Day 14) x 100

Historical control data:

Results from the control group in the current study are acceptable as comparing to historical control data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths in the P generation.
All animals in the treated groups were observed to have blue coloration of the skin and fur throughout the study due to the nature of the test article. All other clinical observations were unremarkable.

- BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Females
During the pre-pairing period, overall mean body weight gains were slightly reduced in the group receiving 1000 mg/kg/day, with statistical significance achieved (P<0.05) between Weeks 5 and 10. In the groups receiving 500 or 1000 mg/kg/day there was a dose-related reduction in
mean body weight gain between Days 7 and 20 of gestation when compared with the controls, with statistical significance achieved at 1000 mg/kg/day (P<0.05, Days 7-14; P<0.01, Days 14-20). At 200 mg/kg/day mean body weight and body weight gains during gestation were similar to the controls. Mean body weight gain for the lactation period was greater in the treated groups, in a dose-related manner, compared with controls and this was significant (P<0.05) in the group receiving 1000 mg/kg/day between Days 7 and 14 of lactation.

- TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There was no effect of treatment on female mean food intake during the pre-pairing period, gestation or lactation. Although there was a statistically significant (P<0.01) increase in food intake in females receiving 500 mg/kg/day during the pre-pairing period, this was not considered to be an adverse effect of treatment.

- REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrous cycle regularity before pairing was unaffected by treatment. The majority of animals mated during the first estrous cycle. In all groups, the pre-coital time was 3 to 3.5 days and there were no adverse effects of treatment on the mating, fertility or fecundity indices. Although fertility and fecundity indices were lower than the controls in the group receiving 1000 mg/kg/day, values were with the background data range

- ORGAN WEIGHTS (PARENTAL ANIMALS)
In females receiving 200, 500 or 1000 mg/kg/day there was a dose-related, statistically significant (P<0.01; P<0.001), increase in mean adrenal weight compared with controls, when corrected for body weight. Mean spleen weights when corrected for body weight in females receiving 1000 mg/kg/day, were significantly increased (P<0.001; P<0.01) when compared with controls. In females at 500 mg/kg/day mean spleen weight was also increased; however statistical significance was not achieved. Females in 1000 mg/kg/day group also had increased liver weight compared with controls, however statistical significance was not achieved. Females receiving 1000 mg/kg/day had a significant increase (P<0.05) in mean uterus weight when compared with controls. Neither of these findings was considered to be of toxicological significance. All other organ weights were unaffected by treatment.

- GROSS PATHOLOGY (PARENTAL ANIMALS)
Necropsy findings for females in the treatment groups showed dark contents in the digestive tract, which was believed to be associated with the physical nature of the test article. There also was a slight increase in the size of the mesenteric lymph nodes in treated females. No other findings at necropsy that were considered to be related to treatment.

- HISTOPATHOLOGY (PARENTAL ANIMALS)
At 200, 500 and 1000 mg/kg/day, in females, microscopic examination showed a dose-related increase in histiocytosis in the mesenteric lymph node, together with cortical hypertrophy/eosinophilia, and foamy macrophages in the adrenal. In females receiving 200, 500 and 1000 mg/kg/day there was an increase in inflammatory cell foci in the adrenal.

There was a difference in the incidence of liver inflammatory cell foci between control females and females dosed at 1000 mg/kg/day and minor intergroup variations in the incidence of other background lesions. However, inflammatory cell foci of the liver and lymphoid hyperplasia of the mandibular lymph node are commonly recorded in the rat. Similarly, agonal congestion/hemorrhage of the mandibular lymph node is a common terminal or post-mortem event. Therefore, these findings were considered unrelated to treatment.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: other:

3

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

4

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- VIABILITY (OFFSPRING)
One male in the control group was sacrificed in extremis in Week 15 because of impaired motility due to a swollen left hind leg, paw and tail and one female in the 200 mg/kg/day group was found dead prior to the start of the pre-pairing period. These deaths were not considered to be related to treatment.

- CLINICAL SIGNS (OFFSPRING)
Throughout the study all animals in the treated groups were observed to have blue coloration due to the nature of the test article.

- BODY WEIGHT (OFFSPRING)
Males
Males in the group receiving 1000 mg/kg/day started the F1 generation phase of the study slightly lighter than the controls and mean body weight gains were significantly lower (P<0.001) than the controls throughout the study. At 500 mg/kg/day mean body weight gain was also significantly (P<0.05) reduced when compared with the controls, with slightly lower absolute body weight throughout the study. Mean absolute body weight was slightly lower than the controls at 200 mg/kg/day, however mean body weight gains were similar to the controls.

Females
Although females in the group given 1000 mg/kg/day started the F1 generation slightly lighter than the controls during the pre-pairing period, mean body weight gains were similar to the controls so that mean absolute body weight remained lower than the controls throughout the study. In the groups receiving 500 or 1000 mg/kg/day mean body weight gains were slightly, but significantly at 1000 mg/kg/day, lower than the controls during gestation, resulting in lower mean absolute body weight on Day 20 of gestation in these groups. At 200 mg/kg/day body weight gains were similar to the controls. Throughout the lactation period mean body weight gains were generally slightly higher than controls in the groups receiving 500 or 1000 mg/kg/day. At 200 mg/kg/day gains were similar to the controls.

- SEXUAL MATURATION (OFFSPRING)
Estrous cycle regularity before pairing was unaffected by treatment. The majority of animals mated during the first estrous cycle. In all groups, the pre-coital time was 2 to 3 days and there were no adverse effects of treatment on the mating, fertility and fecundity indices; values were all within the historical background ranges.
Spermatogenesis, as assessed by measurements of sperm motility, count and morphology, was unaffected by treatment.


- ORGAN WEIGHTS (OFFSPRING)
In males receiving 200, 500 or 1000 mg/kg/day and in females receiving
1000 mg/kg/day, there was a dose-related, statistically significant (P<0.01; P<0.001), increase in mean adrenal weight compared with controls. Mean adjusted weight was slightly increased in females receiving 200 or 500 mg/kg/day, however, statistical significance was not achieved. Mean spleen weights when corrected for body weight were significantly increased in males receiving 500 or 1000 mg/kg/day (P<0.05; P<0.001, respectively) and in females receiving 1000 mg/kg/day (P<0.05) when compared with controls. In males and females given 1000 mg/kg/day, mean liver weight, when corrected for body weight, was slightly greater than the controls, however, statistical significance was not reached. Mean liver weights in males and females receiving 200 or 500 mg/kg/day were unaffected by treatment.

- GROSS PATHOLOGY (OFFSPRING)
Necropsy findings for males and females in the groups receiving the test material showed dark contents of the digestive tract, which can be associated with the physical nature of the test article. In treated males the mesenteric lymph node was increased in size, particularly at 500 or 1000 mg/kg/day. There was also a slight increase in the size of mesenteric lymph nodes in females treated at 500 or 1000 mg/kg/day, but not to the extent seen in the males, with only one animal recorded in the group receiving 500 mg/kg/day. There were no other findings at necropsy that were considered to be related to treatment.


- HISTOPATHOLOGY (OFFSPRING)
At 200, 500 and 1000 mg/kg/day, microscopic examination showed a dose-related increase in histiocytosis in the mesenteric lymph node and foamy macrophages in the adrenal gland. In addition, there was an increase in adrenal cortical hypertrophy/eosinophilia in animals given 500 and 1000 mg/kg/day. There were minor intergroup variations in the incidence of some background lesions. However inflammatory cell foci of the liver and lymphoid hyperplasia of the mandibular lymph node are commonly recorded in the rat. Similarly, agonal congestion/haemorrhage of the mandibular lymph node is a common terminal or post-mortem event. Therefore, in the absence of a dose-response these findings were all considered to be unrelated to treatment.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analysis of test diets

Analysis results show that prepared formulations were homogenous and stable for up to 7 days. Concentrations of copper in test diets prepared at the beginning and end of the P and F1 generations increased with increasing dose. Although the analyses showed variability in copper concentrations, the diets were prepared at the required concentrations, and such variability is considered to reflect the very low levels of copper present in the test article and therefore these variable results are not considered to have had any impact on the integrity of the study.

 

P generation

 

Morbidity and mortality

There were no deaths in the P generation.

 

Clinical observations

All animals in the treated groups were observed to have blue coloration of the skin and fur throughout the study due to the nature of the test article. All other clinical observations were unremarkable.

 

Body weights

Males

In the 1000 mg/kg/day group mean body weight gain was statistically

significantly reduced(P<0.001) throughout the study when compared with the controls. At 500 mg/kg/day mean body weight gains were significantly reduced from Week 10 (P<0.001) when compared with the controls and remained lower until the end of the study. Mean body weight and body weight gains at 200 mg/kg/day were unaffected by treatment; values were comparable with the controls.

Females

During the pre-pairing period, overall mean body weight gains were slightly reduced in the group receiving 1000 mg/kg/day, with statistical significance achieved (P<0.05) between Weeks 5 and 10. In the groups receiving 500 or 1000 mg/kg/day there was a dose-related reduction in

mean body weight gain between Days 7 and 20 of gestation when compared with the controls, with statistical significance achieved at 1000 mg/kg/day (P<0.05, Days 7-14; P<0.01, Days 14-20). At 200 mg/kg/day mean body weight and body weight gains during gestation were similar to the controls. Mean body weight gain for the lactation period was greater in the treated groups, in a dose-related manner, compared with controls and this was significant (P<0.05) in the group receiving 1000 mg/kg/day between Days 7 and 14 of lactation.

 

Food intake

There was no effect of treatment on male or female mean food intake during the pre-pairing period, gestation or lactation. Although there was a statistically significant (P<0.01) increase in food intake in females receiving 500 mg/kg/day during the pre-pairing period, this was not considered to be an adverse effect of treatment.

 

Compound consumption

The following mean intakes were achieved in the P generation:

 

Dose level (mg/kg/day)     200     500     1000

Males pre-pairing               200     498       998

Females pre-pairing            197    496       985

Females gestation               226     540     1061

Females lactation+                     364      925    1685

Mean female                      262      654    1244

+ The mean values were to Day 14 of lactation as subsequent compound consumption was influenced significantly by the offspring eating the test diet.

 

Haematology

In males given 500 or 1000 mg/kg/day, there was a significant increase in mean red blood cell count (P<0.01) and a significant decrease in mean cell volume (P<0.05), mean cell haemoglobin (P<0.01) and red cell distribution width (P<0.05 dose-response) compared to the controls. There was no effect on these parameters in females. There was a slight, but statistically significant (P<0.05) increase in mean activated partial thromboplastin time in males receiving 1000 mg/kg/day. However in females, there was decrease in mean activated partial thromboplastin time with a significant (P<0.05) dose response. This is not considered to be toxicologically significant. In both males and females given 1000 mg/kg/day, total white blood cell count was significantly higher then controls (P<0.001, males; P<0.01, females) due to significant increases in the number of neutrophils (P<0.001). At 500 mg/kg/day the mean neutrophil count was slightly increased in males and females, with statistical significance in the females (P<0.05).

 

Clinical chemistry

At 1000 mg/kg/day in both males and females, there was a statistically significant increase (P<0.001) in mean aspartate aminotransferase and alanine aminotransferase levels when compared with the controls. Mean inorganic phosphorous and total protein were slightly decreased and mean urea and creatinine levels were slightly increased in males receiving 1000 mg/kg/day resulting in slight dose response significances (P<0.05). There were no other findings considered to be of any toxicological significance, although statistical significance was achieved on occasions at 200 or 500 mg/kg/day, there were no dose relationships and these were considered to be due to the large individual animal variation and not due to an effect of treatment.

 

Mating data

Estrous cycle regularity before pairing was unaffected by treatment. The majority of animals mated during the first estrous cycle. In all groups, the pre-coital time was 3 to 3.5 days and there were no adverse effects of treatment on the mating, fertility or fecundity indices. Although fertility and fecundity indices were lower than the controls in the group receiving 1000 mg/kg/day, values were with the background data range

 

Litter data (F1a)

One female from the group receiving 500 mg/kg/day had total embryo-foetal loss and two females from each of the groups receiving 500 or 1000 mg/kg/day showed total litter loss. There were no findings at necropsy that indicated an adverse effect of treatment. There was no effect of treatment on the mean duration of gestation, number of pups born, pup sex ratio or pup survival.

 

Mean pup weight was similar throughout the groups on Day 1. By Day 4 there was a slight but statistically significant (P<0.05) dose related reduction in mean pup weight in the treated groups when compared with the controls. In the groups receiving 500 and 1000 mg/kg/day, mean pup weights were statistically significantly lower for both males and females on Day 7 of lactation (P<0.01) and remained significantly lower than the controls for the remainder of the lactation period. This resulted in a lower weight gain in these groups over the whole lactation period compared to the controls, which was statistically significant (P<0.01) at 1000 mg/kg/day. At 200 mg/kg/day, mean pup weights were similar or slightly lower than the controls, however, mean weight change throughout lactation was slightly greater than the controls.

 

The macroscopic examination of the weanling offspring at necropsy showed no findings indicative of an adverse effect of treatment. Dark contents observed in the gastro-intestinal tract were due to the nature of the test article and was not considered to be an adverse effect of treatment. There was a slight increase in mean adjusted liver weight in the F1a weanlings from the group receiving 500 or 1000 mg/kg/day, which was significant (P<0.05) in females at 500 mg/kg/day. There was no effect in mean liver weight at 200 mg/kg/day. Group mean spleen, thymus and brain weights, both absolute and when adjusted for terminal body weight, were unaffected by treatment.

 

Seminology

There was no adverse effect of treatment on mean sperm count or motility. Although a statistical examination showed a dose-related reduction in mean average path velocity, values were comparable to the background data range. There were slight but statistically significant (P<0.01; P<0.05) increases in the percentage of sperm abnormalities in the groups receiving 500 and 1000 mg/kg/day when compared with controls.

Organ weights

In males receiving 500 or 1000 mg/kg/day and in females receiving 200, 500 or 1000 mg/kg/day there was a dose-related, statistically significant (P<0.01; P<0.001), increase in mean adrenal weight compared with controls, when corrected for body weight. Mean spleen weights when corrected for body weight in males receiving 500, and in males and females receiving 1000 mg/kg/day, were significantly increased (P<0.001; P<0.01) when compared with controls. In females at 500 mg/kg/day mean spleen weight was also increased; however statistical significance was not achieved. In males given 1000 mg/kg/day, mean liver weight when corrected for body weight was significantly higher (P<0.001) than the controls. Females in this group also had increased liver weight compared with controls, however statistical significance was not achieved. In males treated at 500 or 1000 mg/kg/day mean seminal vesicle weight when corrected for body weight was significantly lower (P<0.05) than the controls. Also, females receiving 1000 mg/kg/day had a significant increase (P<0.05) in mean uterus weight when compared with controls. Neither of these findings was considered to be of toxicological significance. All other organ weights were unaffected by treatment.

 

Necropsy and histopathology 

Necropsy findings for males and females in the treatment groups showed dark contents in the digestive tract, which was believed to be associated with the physical nature of the test article. The mesenteric lymph node was increased in size, particularly at 500 or 1000 mg/kg/day in treated males. There also was a slight increase in the size of the mesenteric lymph nodes in treated females but not to the extent seen in the males, with only one animal recorded in the groups receiving 200 or 500 mg/kg/day. No other findings at necropsy that were considered to be related to treatment.

 

At 200, 500 and 1000 mg/kg/day, in males and females, microscopic examination showed a dose-related increase in histiocytosis in the mesenteric lymph node, together with cortical hypertrophy/eosinophilia, and foamy macrophages in the adrenal. In females receiving 200, 500 and 1000 mg/kg/day there was an increase in inflammatory cell foci in the adrenal.

 

There was a difference in the incidence of liver inflammatory cell foci between control females and females dosed at 1000 mg/kg/day and minor intergroup variations in the incidence of other background lesions. However, inflammatory cell foci of the liver and lymphoid hyperplasia of the mandibular lymph node are commonly recorded in the rat. Similarly, agonal congestion/hemorrhage of the mandibular lymph node is a common terminal or post-mortem event. Therefore, these findings were considered unrelated to treatment.

 

F1 generation

 

Morbidity and mortality

One male in the control group was sacrificedin extremisin Week 15 because of impaired motility due to a swollen left hind leg, paw and tail and one female in the 200 mg/kg/day group was found dead prior to the start of the pre-pairing period. These deaths were not considered to be related to treatment.

 

Clinical observations

Throughout the study all animals in the treated groups were observed to have blue coloration due to the nature of the test article.

 

Body weights

Males

Males in the group receiving 1000 mg/kg/day started the F1 generation phase of the study slightly lighter than the controls and mean body weight gains were significantly lower (P<0.001) than the controls throughout the study. At 500 mg/kg/day mean body weight gain was also significantly (P<0.05) reduced when compared with the controls, with slightly lower absolute body weight throughout the study. Mean absolute body weight was slightly lower than the controls at 200 mg/kg/day, however mean body weight gains were similar to the controls.

 

Females

Although females in the group given 1000 mg/kg/day started the F1 generation slightly lighter than the controls during the pre-pairing period, mean body weight gains were similar to the controls so that mean absolute body weight remained lower than the controls throughout the study. In the groups receiving 500 or 1000 mg/kg/day mean body weight gains were slightly, but significantly at 1000 mg/kg/day, lower than the controls during gestation, resulting in lower mean absolute body weight on Day 20 of gestation in these groups. At 200 mg/kg/day body weight gains were similar to the controls. Throughout the lactation period mean body weight gains were generally slightly higher than controls in the groups receiving 500 or 1000 mg/kg/day. At 200 mg/kg/day gains were similar to the controls.

 

Food intake

Mean food intake for males and females during the pre-pairing period was unaffected by treatment. During gestation and lactation mean intake was slightly lower than controls at 1000 mg/kg/day. At 200 or 500 mg/kg/day, mean intake during gestation was unaffected by treatment.

 

Compound consumption

The following mean intakes were achieved in the F1 generation:

Dose level (mg/kg/day)     200     500     1000

Males pre-pairing               214     531     1044

Females pre-pairing            206     518     1026

Females gestation               217     557     1024

Females lactation+                     317      812     1542

Mean female                       246     629     1198

+ The mean values have been limited to Day 14 of lactation as subsequent compound

consumption was influenced significantly by the offspring eating the test diet.

 

Learning ability

There was no adverse effect of treatment at 1000 mg/kg/day on escape times and numbers of correct escapes in the swimming maze trials.

 

Physical development

There was no adverse effect of treatment on time of balano-preputial separation or vaginal opening.

 

Functional observations

There was no effect of treatment at 1000 mg/kg/day on functional or behavioural development.

 

Locomotor activity

Males and females in the group receiving 1000 mg/kg/day were generally more active than the controls with statistical significance on occasions and overall in the females (P<0.05). However, values were within our background data ranges and due to the large individual animal variation and that no effects of treatment were observed in functional observation battery, this was considered not to be an adverse

effect of treatment.

 

Mating data

Estrous cycle regularity before pairing was unaffected by treatment. The majority of animals mated during the first estrous cycle. In all groups, the pre-coital time was 2 to 3 days and there were no adverse effects of treatment on the mating, fertility and fecundity indices; values were all within the historical background ranges.

 

Litter data (F2a)

There were 3, 3, 7 and 7 females in the groups receiving 0, 200, 500 or 1000 mg/kg/day which had total litter loss. There were no findings at necropsy of these animals that indicated an adverse effect of treatment. There was no effect of treatment on the mean duration of gestation, number of pups born, pup sex ratio or pup survival. Mean pup weight was slightly lower than controls in a dose-related manner throughout the groups on Day 1. By Day 4 pups in the group receiving 1000 mg/kg/day were significantly (P<0.01) lighter than controls and remained so

throughout lactation. Mean weights were lower than control in the groups receiving 200 or 500 mg/kg/day, with statistical significance achieved on occasions. Mean percent weight gain during lactation was significantly lower at 1000 mg/kg/day (P<0.01) and slightly lower at 500 mg/kg/day, although not statistically significant. Weight change in the group receiving 200 mg/kg/day was slightly greater than the controls. The macroscopic examination of the weanling offspring at necropsy showed no findings indicative of an adverse effect of treatment. Dark contents of the gastrointestinal tract was expected due to the nature of the test article and was not considered to be an adverse effect of treatment. Group mean spleen, liver, thymus and brain weights, both absolute and when adjusted for terminal body weight, of the F2a weanlings were unaffected by treatment.

 

Seminology

Spermatogenesis, as assessed by measurements of sperm motility, count and morphology, was unaffected by treatment.

 

Organ weights 

In males receiving 200, 500 or 1000 mg/kg/day and in females receiving

1000 mg/kg/day, there was a dose-related, statistically significant (P<0.01; P<0.001), increase in mean adrenal weight compared with controls. Mean adjusted weight was slightly increased in females receiving 200 or 500 mg/kg/day, however, statistical significance was not achieved. Mean spleen weights when corrected for body weight were significantly increased in males receiving 500 or 1000 mg/kg/day (P<0.05; P<0.001, respectively) and in females receiving 1000 mg/kg/day (P<0.05) when compared with controls. In males and females given 1000 mg/kg/day, mean liver weight, when corrected for body weight, was slightly greater than the controls, however, statistical significance was not reached. Mean liver weights in males and females receiving 200 or 500 mg/kg/day were unaffected by treatment.

 

Necropsy and histopathology

Necropsy findings for males and females in the groups receiving the test material showed dark contents of the digestive tract, which can be associated with the physical nature of the test article. In treated males the mesenteric lymph node was increased in size, particularly at 500 or 1000 mg/kg/day. There was also a slight increase in the size of mesenteric lymph nodes in females treated at 500 or 1000 mg/kg/day, but not to the extent seen in the males, with only one animal recorded in the group receiving 500 mg/kg/day. There were no other findings at necropsy that were considered to be related to treatment. 

 

At 200, 500 and 1000 mg/kg/day, microscopic examination showed a dose-related increase in histiocytosis in the mesenteric lymph node and foamy macrophages in the adrenal gland. In addition, there was an increase in adrenal cortical hypertrophy/eosinophilia in animals given 500 and 1000 mg/kg/day. There were minor intergroup variations in the incidence of some background lesions. However inflammatory cell foci of the liver and lymphoid hyperplasia of the mandibular lymph node are commonly recorded in the rat. Similarly, agonal congestion/haemorrhage of the mandibular lymph node is a common terminal or post-mortem event. Therefore, in the absence of a dose-response these findings were all considered to be unrelated to treatment.

Applicant's summary and conclusion

Conclusions:

Dietary administration of 500 and 1000 mg/kg/day of the test substance to rats for two successive generations produced adult toxicity in terms of lower body weight gain. Also, at 500 and 1000 mg/kg/day, pup growth was impaired in both generations. The NOAEL based on reduced body weight gain in adults and pups is 200 mg/kg/day.

In both generations, the no-observed-adverse-effect-level (NOAEL) for reproductive parameters was 1000 mg/kg/day.

The statistically significant increase in white blood cells at 1000 mg/kg/day, and in lymphocytes and neutrophils at 500 and 1000 mg/kg/day combined with a dose-related increase in adult mean adrenal weight with a dose-related increase in cortical hypertrophy/eosinophilia, foamy macrophages and/or inflammatory cell foci and a dose-related increase in histiocytosis of the mesenteric lymph node in both generations (200, 500 and 1000 mg/kg/day) suggest an immune response to the test material.

Executive summary:

A two generation Reproduction study was conducted with to evaluate the potential to cause effects on gonadal function, the estrous cycle, mating behavior, conception, pregnancy, parturition, lactation and weaning, and the growth and development of the offspring when administered orally, by diet. Groups of 24 male and 24 female parental rats (P generation) were fed diets containing 0, 200, 500 or 1000 mg/kg/day of the test material that was verified analytically. The parental (P) animals were fed the test diets for ten weeks before pairing and until necropsy. The P females were allowed to litter and rear their offspring (F1a) to weaning. Twenty-four animals of each sex of the offspring were randomly selected from each group to form the main study filial (F1) generation. Direct treatment of the F1 generation commenced at weaning and continued for ten weeks post-weaning (maturation phase), pairing and until necropsy. All main study F1 females were allowed to litter and rear their offspring (F2a) to weaning. All animals were observed for clinical signs and mortality. Body weights, food intake, hematology, clinical chemistry, estrous cycle, mating, litter data, physical development, functional and behavior development, organ weights and gross organ and microscopic tissue evaluations were conducted.

 

P generation

 

There was no mortality and only clinical observation was a blue coloration in the treated groups throughout the study due to the test article. 

 

At 500 and 1000 mg/kg/day, mean body weight gain was reduced when compared with the controls. There was no effect of treatment on male or female mean food intake.

 

In males given 500 or 1000 mg/kg/day, mean red blood cell count was significantly increased with significantly lower mean cell volume, mean cell hemoglobin and red cell distribution width. There was a significant increase in neutrophil and total white blood cell count in males and females receiving 1000 mg/kg/day. At 500 mg/kg/day mean neutrophil count was slightly increased in males and females.

 

At 1000 mg/kg/day in males and females, there was a statistically significant increase in mean aspartate aminotransferase and alanine aminotransferase levels when compared with the controls. Mean inorganic phosphorous and total protein was slightly decreased and mean urea and creatinine was slightly increased in males receiving 1000 mg/kg/day.

 

Estrous cycle regularity before pairing was unaffected by treatment and there were no adverse effect of treatment on the mating, fertility and fecundity indices. There was no effect of treatment on the mean duration of gestation, number of F1a pups born, pup sex ratio or pup survival. There was a slight, but statistically significant, dose-related reduction in mean pup weight in the groups receiving 500 or 1000 mg/kg/day. At 200 mg/kg/day, mean pup weights were similar to, or slightly lower than, the controls. The macroscopic examination of the weanling offspring at necropsy showed no findings indicative of an adverse effect of treatment.

There was a slight increase in mean adjusted liver weight in the F1a weanlings from the group receiving 500 or 1000 mg/kg/day. There was no effect on mean liver weight at 200 mg/kg/day. Group mean spleen, thymus and brain weights, both absolute and when adjusted for terminal body weight, were unaffected by treatment.

 

There was no adverse effect of treatment on parental males mean sperm count or motility. There was a slight but statistically significant increase in sperm abnormalities in the groups receiving 500 and 1000 mg/kg/day when compared with controls.

 

In P males receiving 500 and 1000 mg/kg/day, and in P females receiving 200, 500 and 1000 mg/kg/day, there was a statistically significant increase in mean adrenal weight. Mean spleen weights in males and females receiving 500 and 1000 mg/kg/day were increased when compared with controls. In males and females given 1000 mg/kg/day, mean liver weight when corrected for body weight was increased compared with the controls. In males given 500 or 1000 mg/kg/day, mean seminal vesicle weight, when corrected for body weight, was significantly lower than the controls. Females receiving 1000 mg/kg/day had a significant increase in mean uterus weight when compared with controls. All other organ weights were unaffected by treatment.

 

At 200, 500 and 1000 mg/kg/day, in parental males and females, microscopic examination showed a dose-related increase in histiocytosis in the mesenteric lymph node, together with cortical hypertrophy/eosinophilia, and foamy macrophages in the adrenal. In

females receiving 200, 500 and 1000 mg/kg/day there was an increase in

inflammatory cell foci in the adrenal.

 

F1 generation

 

There was no treatment-related mortality, and the only clinical observation was a blue coloration in the treated groups throughout the study due to the test article. 

 

At 500 and 1000 mg/kg/day, mean body weight gain was reduced when compared with the controls. There was no adverse effect of treatment on mean food intake during the pre-pairing period. During gestation and lactation mean intake was slightly lower than controls at 1000 mg/kg/day.

There was no adverse effect of treatment on learning ability, physical development or on functional or behavioural development.

Males and females in the group receiving 1000 mg/kg/day were generally more active than the controls in the motor activity recorder.

 

Estrous cycle regularity before pairing was unaffected by treatment and there were no adverse effects of treatment on the mating, fertility and fecundity indices. There was no effect of treatment on the mean duration of gestation, number of F2a pups born, pup sex ratio or pup survival.

Mean pup weight was slightly lower than controls in a dose related manner (P<0.05) throughout the groups on Day 1post-partum. Weight gain during lactation was significantly (P<0.01) lower than control at 1000 mg/kg/day. The macroscopic examination of the weanling offspring at necropsy showed no findings indicative of an adverse effect of treatment. Group mean spleen, liver, thymus and brain weights, both absolute and when adjusted for terminal body weight, of the F2a weanlings were unaffected by treatment.

 

Spermatogenesis, as assessed by measurements of sperm motility, count and morphology in the F1 animals, was unaffected by treatment. 

 

Mean adrenal weight was increased in the treated groups when compared with the controls. Mean spleen weights were significantly increased in males receiving 500 and 1000 mg/kg/day and in females receiving 1000 mg/kg/day. In males and females given 1000 mg/kg/day, mean liver weight was slightly higher than the controls.

 

At necropsy, in males and females given 500 or 1000 mg/kg/day, the mesenteric lymph node was increased in size. At 200, 500 and 1000 mg/kg/day, microscopic examination showed a dose-related increase in histiocytosis in the mesenteric lymph node and foamy macrophages in the

adrenal gland. In addition, there was an increase in adrenal cortical

hypertrophy/eosinophilia in animals given 500 and 1000 mg/kg/day.

 

In conclusion, dietary administration of 500 and 1000 mg/kg/day of the test substance to rats for two successive generations produced adult toxicity in terms of lower body weight gain. Also, at 500 and 1000 mg/kg/day, pup growth was impaired in both generations. The NOAEL based on reduced body weight gain in adults and pups is 200 mg/kg/day.

 

In both generations, the no-observed-adverse-effect-level (NOAEL) for reproductive parameters was 1000 mg/kg/day.

 

The statistically significant increase in white blood cells at 1000 mg/kg/day, and in lymphocytes and neutrophils at 500 and 1000 mg/kg/day combined with a dose-related increase in adult mean adrenal weight with a dose-related increase in cortical hypertrophy/eosinophilia, foamy macrophages and/or inflammatory cell foci and a dose-related increase in histiocytosis of the mesenteric lymph node in both generations (200, 500 and 1000 mg/kg/day) suggest an immune response to the test material.