Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17.9.2008-19.12.2008
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
B.31, Council Regulation (EC) No.440/2008, Published in O.J. L142,2008
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material:Dusantox L - liquid viscous
Chemical name: 448-020-2 N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine, reaction products with 2-phenylpropene

Composition:
1. N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine
61,06 % area
CAS No.: 793-24-8
Molecular formula:C18H24N2
Molecular weight: 268,4
Smiles Code:N(c(ccc(Nc(cccc1)c1)c2)c2)C(CC(C)C)C

2.N-[4-(alpha,alpha'dimethylbenzyl)phenyl]-N'-(1,3-
dimethylbutyl)-p-phenylenediamine (i.e. p-cumyl-6PPD)
31,28 % area
CAS No.: 194478-84-7
Molecular formula:C27H34N2
Molecular weight: 386,6
Smiles Code:CC(C)CC(C)(Nc1ccc(Nc2ccc(C(C)(C)c3ccccc3)cc2)cc1)

o-cumyl-6PPD 2,38 % area

cumyl-6PPD (other) 2,84 % area

3. Impurities
alpha-methylstyrene 0,01 % area
CAS No.: 98-83-9
dimers of alpha-methylstyrene 2,09 % area

Batch. No:002/2008

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Sex animals: sexually adult females (males only for mating)
Age at start of the study: 9 weeks
Acclimatization: 6 daye
Total number of animals: 100 females (25 males)
Housing:
Animals were housed in SPF animal room in plastic cages containing sterilised clean shavings of soft wood. Before mating 2 rats of the same sex in
one cage, during mating period-one male and two females in one cage were housed. During pregnancy 1 female was housed in one cage.
Selection of females:
Pregnant females were randomly selected to the experimental groups after determination of pregnancy.
Identification:
Identification of females was made by colour marks on fur (system 1-10), each cage was marked with the number of study, number of animals, sex,
number of cage, name and dose of the test substance, mark of group and date of planned euthanasia.
Diet:
Complete peleted diet for rats and mince in SPF breening was used, manufacturer: Ing.Miroslav Mrkvička, Jesenice u Prahy. Diet was sterilised befor
using. Composition of food: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted groats, Wheat sprout, dehydrated yeast, Calcium
carbonate, Vitamin and Mineral complex.
Water:
Free access to drinking water (water ad libitum). Water quality corresponded to regulation No.252/2004 Czech Coll. of Law, Health Ministry. Water
was sterilised before using.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
olive oil
Details on exposure:
Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with males. The test substance was then
administered to pregnant females - daily from the 5th to the 19th day of pregnancy. The study included four groups of females - 3 treated groups
and 1 control group (vehicle only). The test substance was administered suspended in olive oil by stomach tube and the concentrations of
suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight.
The treated and control females were administered daily by gavage - from the 5th to the 19th day of pregnancy. Oral way of administration was
chosen on the request of sponsor. The animals were treated 7 days per week at the same time (8.00 - 10.00 am).The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form (test substance suspension in
olive oil) was prepared daily just before administration. The vehicle control group was administered by olive oil in the same volume. The application
form of the test substance was prepared daily before administration, these suspensions were mixed for 10 minutes by magnetic stirrer. The
procedure was based on the results of analyses of test substance application form homogeneity and stability (Annex 1 of the Study No.27/08/8:
Dusantox L - repeated dose 90-day Oral Toxicity study, VIUOS-CETA Report No.0988,2009).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose levels for study:
25 mg/kg/day
50 mg/kg/day
100 mg/kg/day were chosen on the basis of results of study No. 27/08/8 :Dusantox L - repeated Dose 90-day Oral Toxicity study, VUOS-CETA
Report No. 0988,2009.
Details on mating procedure:
After acclimatisation females were mated with males (1 male and 2 females). Vaginal smears were carried out daily in the morning to control
fertilization (first time: 24 hours after the first removing to male). Presense of sperms was examined. Day 0 of pregnancy was the day on which
sperms in vaginal smears were observed. Pregnant females were randomly distributed to experimental groups - 24 or 25 probably gravid females
were at each group (2 females from beginning total number 100 were not used for study).
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily from the 5th to the 19th day - 1 mL per 100 g of body weight
Duration of test:
Test duration: 20 days
Doses / concentrations
Remarks:
Doses / Concentrations:
25 ,50 and 100 mg/kg body weight
Basis:
nominal conc.
25 mg/kg of body weight
No. of animals per sex per dose:
The dose levels were determined on the basis of results of study No. 27/08/8 :Dusantox L - Repeated Dose 90-day Oral Toxicity study, VUOS-CETA Report No. 0988,2009.
1. Control: 25 probably pregnant females No. 101-125
2. 25 mg/kg 24 probably pregnant females No. 126-149
3. 50 mg/kg 24 probably pregnant females No. 151-174
4. 100 mg/kg 25 probably pregnant females No. 176-200
Note: numbers 150,175 - were not assigned (only 24 probably pregnant females were used for study at each dose level).
Control animals:
yes, concurrent vehicle
Details on study design:
Objective of study: The goal of this study was Prenatal developmental toxicity of
Dusantox L in rats.

Examinations

Maternal examinations:
Health condition control of females:
No negative changes of health condition were found out in females of the control group and at the lowest dose during whole study. Only one female with vaginal discharge was recorded at the lowest dose.
At the middle dose level, in 7 females apathy only was recorded in the last week of pregnancy.In females of the highest dose level clinical symptoms
of toxicity were observed from the 4th day of application to the end study. In the 2nd week of study the following clinical symptoms of toxicity were
detected: Piloerection in 21 females, difficult application in 13 females, gibbous pose in 15 females, cachexia in 9 females, red secreta around
nostrils or eyes in 11 females, apathy in 4 females, anemia in 8 females, overgrown teeth in 8 females and ataxia in 1 female.
In the last week of study the health condition of all females was not good: piloerection in all females, difficult application in 19 females, gibbous pose
in 23 females, cachexia in 9 females, red secreta around nostrils or eyes in 23 females, apathy in 19 females, anemia in 17 females, overgrown teeth
in 17 females, tachypnoe in one female and ataxia in 4 females were recorded. (See Table 4 of this study)

Clinical observation of females:
No clinical symptoms of intoxication were observed at control group and at the lowest dose level.
In females of the middle dose level negative clinical changes were observed sporadically: red secreta around nostrils or eyes were detected in 2
females and piloerection in one female were recorded at the 2nd week. Similar findings were found out at the 3rd week: red secreta in 4 females and
piloerection in 3 females.
In females of the highest dose level clinical symptoms of toxicity were observed in the second and third week of study. In the 2nd week secreta
around nostrils or eyes in 10 females, piloerection in 20 females, caxhexia in 12 females, gibbous pose in 11 females and anemia in 14 females. Only one female shoved ataxia, apathy, dyspnoea or vocalisation.
Similar findings were found out at the 3rd week: red secreta around nostrils or eyes (in 16 females), piloerection (in all females), gibbous pose (in 16 females), cachexia (in 21 females),ataxia (in 4 females), anemia ( in 17 females), apathy (in 11 females) and vocalisation (in 3 females) were recorded. Dyspnoea or tachypnoea occured in only 1 female. (See Table 5 of this study)

Pathological examination of females:
biometry of uterus: only females who were found pregnant on the 20th day of gravidity (females with foetuses) were used for calculation of average
values. Decreases of absolute weights of uterus were recorded at all treated groups (statistically significantly at the highest dose level). Decrease of
relative weights was found out in treated females at the lowest and middle dose levels. Slightly increased relative weight of uterus was recorded at the highest dose level. ( See Table 6 of this study)

macroscopic examination: macroscopic changes were evaluated in all females (including females without foetuses). No macroscopic finding was
recorded at the control group and at the lowest dose level. At the middle dose level only haemorrhages on stomach mucosa, overgrown teeth or
haemorrhage on amnion were recerded in 1 female. At the highest dose level macroscopic findings were diagnosed in 21 females - mainly in
digestive system. 19 females showed overgrown teeth. In stomach the following changes were described: erosions of mucosa in 12 females, chyme
with blood in 2 females, ulceration of mucosa in 2 females, oedematous mucosa in 1 female and congested mucosa in 1 female. Chyme with blood in
intensines was recorded in 2 females. Local changes (whitish punctiform foci) in lungs or haemorrhages in heart were found out in one female.
Reduced spleen was recorded in 4 females. 8 females showed red secreta around nostrils or eyes. Haemorrhages on amnion in 1 female were found out in reproductive tract. ( See Table 7 of this study)


Ovaries and uterine content:
Reproduction parameters:
Intra Uterine Death Early and Intra Uterine Death Late- for evalution of IUDE and IUDL females without foetuses and without implantations were not
used.Number of corpora lutea in females were well-balanced in control and treated groups. Slight differences were detected in number of
implantations and resorptions. The highest frequence of resorptions was in females at the highest dose level. Preimplantation losses (IUDE) in
treated females were higher than in control females. The highest difference was recorded at the middle and lowest dose levels. Postimplantation
losses (IUDL) were analogous in control and in females at the lowest dose levels. Increased preimplantation losses were recorded in females treated
by the test substance at the middle dose level and more marked the highest dose level.
(See Table 8 of this study) - Parameters of reproduction
(See Table 9 of this study) - IUDE and IUDL (% per female, average)
Fetal examinations:
Number of foetuses:
Only females who were found pregnant on the 20th day of gravidity (females with foetuses) were used foe calculation of average values. Average
total number of foetuses in litter, number of male and female foetuses in females of the highest and middle dose levels was analogous with control.
Total number of foetuses (in litter) in females of the lowest dese level was slightly decreased against control. Also number of male and female
foetuses was accrdingly changed in the lowest dose level: number of male foetuses and number of female foetuses were slightly decreased compared to control. Sex ratio was without change. Statistically significant differences were not detected.
(See Table 10 of this study) - number of foetuses in litter (averages)

Pathological examinatin of foetuses:
Body weight of foetuses-decreased average body weight of foetuses with dependence on the dose level was recorded at all dose levels (in
comparison with the control group). Also average body weights of male foetuses and female foetuses of females treated by the test substance were
lower than at the control females (also with dependence on the dose). Statistically signifocant decrease of weight was found out at the highest dose
level.
(See Table 11 of this study) - Body weight of foetuses (grams, averages)

Examination of external alternations - during examination of symmetry of fore and hind limbs, number of fingers, closing opening of eye fissures
and external auditory canal, symmetry of head, integrity of superior palatum, status of umbilicus and genital papilla no serious alternations were
often observed. In foetuses of treated females only small finding without dependence on dose level was rarely recorded. Foetuses with haemorrhages in subcutis were observed sporadically in a few litters of control and treated females.
(See Table 12 and 13 of this study)

Examination of internal alterations-soft tissues: during careful gross dissections of foetuses placing and morphology of organs and big vessels was reviewed. In foetuses of females from the control group no findings were recorded. Affected foetuses were rarely found out at all treated groups. At the lowest dose level clouded humour in abdominal cavity was found out in one foetus. Blood clots on liver of two foetuses and blood clots in abdominal cavity of two foetuses were recorded at the middle dose level. At the highest dose level overgrown forelimbs in two foetuses were observed. Other findings were not detected.
(See Table 14 of this study)- Macroscopic changes of soft tissues - individual foetuses
(See Table 15 of this study)- Macroscopic changes of soft tissues - litters

Examination of internal alterations-skeleton: During examination of foetal cranium unossifified (or absented) supraoccipital bone was found out in 12 foetuses at the highest dose level and in one foetus at the lowest dose level. At the highest dose level this change was found out in the 28.6 % of litters. Incomplete ossification of supraoocipital bone was recorded also only at the lowest and highest dose levels and affected c. 9 % foetuses (more than 20 % of litters). Incomplete ossification of parietal bone and interparietal bone were recorded in all treated groups. Percentual portion of affected foetuses and portion of litters with affected foetuses was increased with dependence on the dose level. Incomplete ossification of frontal bone was recorded at the middle dose level (3 foetuses) and at the highest dose level (in 8 foetuses). During examination of foetal skeleton incomplete ossification of sternum was recorded in foetuses of all groups. Percentual portion of foetuses with this variation was markedly increased in treated groups compared to control and this difference was dependent on dose level. At the highest dose level almost all litters were affected. No variations of ossification of vertebrae were recorded at the control group and at the middle dose level. Changes were sporadically recorded at the lowest dose level (incomplete ossification of cervical vertebrae in 4 foetuses and unossified sacral vertebrae in 5 foetuses). On the contrary hight incidence of variations was recorded at the highest dose level. Incomplete ossification of cervical vertebrae and unossified sacral vertebrae were found out in more than 40 % of foetuses (more than 50 % of litters). Unossified lumbar vertebrae (absence of ossification sites), incomplete ossification of lumbar or sacral vertebrae were recorded also only at the highest dose level. Anomaly of ribs-undulation along the lenght of a rib was detected in foetuses of the control group and at the lowest and at the highest dose levels. The highest percentual portion of litters with affected foetuses was at the control group. Percantual portion of affected foetuses at all affected groups was under 10 %.
(See Table 16 of this study)-Skeletal alterations (number of affected foetuses)
(See Table 17 of this study)-Skeletal alterations (% portion of affected foetuses)
(See Table 18 of this study)-Skeletal alterations (number of litters with affected foetuses)
(See Table 19 of this study)-Skeletal alterations (% portion of litters with affected foetuses)
Statistics:
The ANOVA test - Analysis of variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight of pregnant females at the end of pregnancy, absolute and relative weight of uterus, number of foetuses, number of male foetuses, number of female foetuses, weight of foetuses,, weight of male foetuses and weight of female foetuses. control group with vehicle was compared with three treated groups.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of averages.
Indices:
Reproduction parameters:
In uterus number of viable foetuses, number of dead foetuses and number of resorptions (implantation without recognisable embryo/foetus or dead embryo or foetus with external degenerative changes) were recorded. Number of corpora lutea on ovaries was found out.
Preimplantation and posimpantation losses were calculated from number of implantations (=number of foetuses plus number of resortions),
corpora lutea and resorptions were also couted.

Preimplantation loss - IUDE (Intra Uterine Death Early):


Corpora lutea – implantations x 100
Corpora lutea

Postimplantation loss - IUDL (Intra Uterine Death Late):

resorptions x 100
implantations

Uteri of non-pregnant females were examined by the help of ammonium sulphide staining to confirm the non-pregnant status (female who did not
become pregnant) or to find out empty implantations sites (female who became pregnant and then aborted).
Historical control data:
The procedure was based on the results of analyses of test substance application foer homogeneity and stability (Anex 1 of the Study No. 27/08/8, Dusantox L - Repeated Dose 90-day Oral Toxicity study, VUOS-CETA Report No. 0988,2009).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Developmental effect of the test substance along with toxicity to the maternal animals were detected at the highest dose levels. At the middle and
lowest dose level the developmental effcte (decreased weight of foetuses, the increased incidence of skeletal variations) occurred in the absence of
toxicity to the maternal animals.
The test substance had negative effect on growth of maternal animals. Body weight of maternal animals at the highest dose level was significantly decreased in comparison with the control animals from the 7th day of application to the end of study. At the end of application marked tabescence in this group was recorded. Cachexia of some animals was also recorded. The body weight of the lowest and middle dose levels was relatively well-balanced with the control group.Food consumption of treated maternal animals was lower than at the control from the 8th day to the end of study. The difference was more marked at the highest dose level.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
< 25 mg/kg bw/day
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
In individual examination of the foetuses decrease of the weight was found especially at the highest dose level. Increased number of aborted females and related negative changes of reproduction parameters (postimplantation losses) were also recorded in treated groups with higher incidence at
highest and middle dose levels. The significant incidence of skeletal malformation was recorded at the highest dose level. The increased incidence of some of skeletal variations was found out in foetuses of all treated groups and could be considered as an adverse effect of treatment.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Results: At the dose level of 25 mg/kg/day the weight incremets, mortality, health condition, clinical status after application, macroscopical structure of organs of pregnant females, some values of reproduction parameters (uterus biometry, number of dead foetuses, intra uterine death late), development of foetuses (weight of foetuses, evaluation of external and visceral affections) were unaffected by treatment of the test substance. At the dose level of 50 mg/kg/day the weight increment, health condition, mortality, clinical status after application, macroscopical structure of organs of pregnent females, some values of reproduction parameters (uterus biometry, number of live and deat foetuses), development of foetuses (evaluation of external and visceral affections) were unaffected by tretment of the test substance. At the dose level 100 mg/kg/day the mortality, reproduction parameter (number of live foetuses, intra uterine death early) of pregnant females and foetal development judged according to the external and visceral observation were unaffected by tretment of the test substance. The following effects could be attributed to the administration of the test substance: At the dose level of 25 mg/kg/day number of live foetuses decrease was very slightly decreased. Increased incidence of delayed ossification of vertebrae, sternum and cranium bones (skeletal variations) was recorded. At the dose level of 50 mg/kg/day the following effects were observed: increased intra uterine death early and late, decrease in number of live foetuses, slight decrease in foetus weight, increases incidence of skeletal variation (delayed ossification of sternum and cranial bones). At the dose level of 100 mg/kg/day significantly decreased body weight increment, markedly decreased food consumption, occurrence of clinical symptoms of toxicity, significant decrease of absolute weight and changes of macroscopical structure of organs in maternal animals (findings in digestive system) were influenced by administration of the test substance. Important effect on reproduction parameters (high incidence of aborted females, high incidence of resorptions and accompanying high postimplantation losses, incidence of dead foetuses), weight of male and female foetuses (significantly decrease) and presence of skeletal malformations (unnosified or absented supraoocipital bone) and high incidence of skeletal variation (delayed ossification of vertebrae, sternum and cranium bones) were also recorded.

Applicant's summary and conclusion

Conclusions:
The administration of the test substance Dusantox L affected growth,clinical status and macroscopic structure of organs in treated maternal
animals. These effects were significantly manifested in the results of health condition controls and clinical observations (cachexia, anemia, secretion from nostrils and eyes, piloerrection, apathy). The weight of pregnant females was significantly decreased. During the necropsies of mothers the
patologic changes in stomach and significantly lower absolute weight of pregnant uterus were found. Above mentioned changes were recorded at
the highest dose level. In treated groups, increase in postimplantation losses - increased number of aborted females were recorded in treated
groups with higher incidence at highest and middle dose levels. In individual examination of the foetuses decrased weight was found out especially
at the highest dose level.
The significant incidence of skeletal malformation was recorded at the highest dose level. The dose-dependent increased incidence of some of
skeletal variations in comparison with the control group was found out in foetuses of all treated groups and could be considered as an adverse effect of treatment.
In individual examination of the foetuses decrease of the weight was found especially at the highest dose level. Increased number of aborted females and related negative changes of reproduction parameters (postimplantation losses) were also recorded in treated groups with higher incidence at
highest and middle dose levels.
Executive summary:

Developmental effect of the test substance along with toxicity to the maternal animals were detected at the highest dose levels. At the middle and lowest dose level the developmental effects (decreased weight of foetuses, the increased incidence of skeletal variations) occurred in the absence of toxicity to the maternal animals.

Test batch No.: 002/2008