Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol, 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2016 - September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(4-methylphenyl)imino]bisethanol

EC Number:

221-359-1

EC Name:

2,2'-[(4-methylphenyl)imino]bisethanol

Cas Number:

3077-12-1

Molecular formula:

C11H17NO2

IUPAC Name:

2-[(2-hydroxyethyl)(4-methylphenyl)amino]ethan-1-ol

Constituent 2

Reference substance name:

2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethan-1-ol

Cas Number:

878391-30-1

Molecular formula:

C13H21NO3

IUPAC Name:

2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethan-1-ol

Constituent 3

Reference substance name:

Higher ethoxylated p-toluidine derivative-1

IUPAC Name:

Higher ethoxylated p-toluidine derivative-1

Constituent 4

Reference substance name:

Higher ethoxylated p-toluidine derivative-2

IUPAC Name:

Higher ethoxylated p-toluidine derivative-2

impurity 1

Test material form:

liquid: viscous

Details on test material:

- Physical appearance: Clear slightly yellowish to brown viscous liquid
- Storage conditions: At room temperature

Specific details on test material used for the study:

May partially solidify if cooled below 20°C and/or if dust or seeding crystals are present. The sample may be heated to approximately 30°C to dissolve the solid.

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: approximately 10-14 weeks
- Weight at study initiation: 168 - 263 grams
- Fasting period before study: no
- Housing: Individually in Macrolon plastic cages (MIII type); Sterilized sawdust as bedding material and paper as cageenrichment/ nesting material were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: Free access to tap-water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09 August 2016 To: 01 september 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item (1.113) and vehicle (1.036). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method (Test Facility Study No. 501370). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Details on mating procedure:

Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 postcoitum was the day of successful mating; confirmed by vaginal plug).

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive.

Frequency of treatment:

Once daily for 7 days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

22 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on results of the dose range finding study

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: according to Guidelines

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
• Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites)/ number of corpora lutea) x100

Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/ number of implantation sites) x100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%) = (number of viable fetuses affected/litter/ number of viable fetuses/litter) x 100

Historical control data:

included in the report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Remarkable signs of toxicity were observed for one female at 600 mg/kg. This female showed rales from Day 7 post-coitum onwards and piloerection on Days 9-12 and Days 17-20 post-coitum. Moreover, smacking was noted on Days 8-16 post-coitum. Lethargy, salivation and hypothermia were observed for the last four, two and one day(s) of the observation period, respectively. A treatment relation of these observations in a single female is unlikely but cannot be excluded. As the combination of these clinical signs were observed for one single female, this female was excluded from the summary tables because it was considered to be an outlier.
For the remaining females in this study the following clinical signs of toxicity were noted. Rales was observed for four females at 600 mg/kg bw/day, compared to one female at 200 mg/kg bw/day. The finding in the female at 200 mg/kg bw/day was not considered to be toxicologically significant, as it was noted for one single mid dose female for only three days.
Tremor and lethargy were noted both for three females at 600 mg/kg bw/day on Day 6 post-coitum (i.e. the first day of dosing) and Days 6-8 post-coitum. The findings in the 600 mg/kg bw/day females were considered to be treatment related.
Incidental findings that were noted included alopecia and chromodacryorrhoea. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. Consequently, these findings were considered signs of no toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female of the 600 mg/kg bw/day group was found dead on Day 20 post-coitum before treatment. The tonic spasms were observed directly after dosing and recovered after two minutes. Body weights and food consumption were considered to be normal and at necropsy, no macroscopic findings were noted. This female was pregnant and had 13 fetuses, of which no external abnormalities were noted. The reason for this spontaneous death could not be identified, but a relationship to treatment cannot be excluded.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly lower body weights and statistically significantly lower body weight gain were noted at 600 mg/kg bw/day from Day 9 post-coitum onwards. In addition at this dose, weight gain corrected for gravid uterus was statistically significantly lower as compared to the concurrent control group (13.3 gram versus 24.7 gram).
At 200 mg/kg bw/day, body weight gain was statistically significantly lower on Days 12 and 15 postcoitum.
Recovery occurred during the remainder of the treatment period. Weight gain corrected for gravid uterus remained in the same range as controls.
No toxicologically relevant changes in body weights, body weight gain or weight gain corrected for gravid uterus were noted by treatment at 60 mg/kg bw/day.
One high dose female, which showed remarkable signs of toxicity (see description in the clinical signs section), had significantly lower body weights and body weight gain (corrected for gravid uterus) during
the entire observation period. A body weight loss of 20% was noted on Day 9 post-coitum. Signs of recovery were noted the days after, as her body weight was slightly increased on Days 15 and 18 post-coitum. A body weight gain corrected for gravid uterus of -57 gram was noted. This female was excluded from the summary body weight tables.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption before and after correction for body weight was statistically significantly reduced at 600 mg/kg bw/day from Day 6 to Day 15 post-coitum.
At 200 mg/kg bw/day, lower food consumption was noted on Day 6 to Day 12 post-coitum. This was not statistically significant for relative food consumption on Days 6-9 post-coitum.
Treatment at 60 mg/kg bw/day did not result in any toxicologically relevant changes in food consumption.
The high dose female with severe clinical signs as described above had a significantly lower food consumption on Days 6-12 post-coitum when compared to the other females of the 600 mg/kg bw/day group. This female was excluded from the summary food consumption tables.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 200 and 600 mg/kg bw/day, liver weights relative to body weights were statistically significantly increased with 6% and 13% when compared to controls, respectively.
At 60 mg/kg bw/day, liver weights were similar to control values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment related macroscopic findings were seen in one female at 600 mg/kg bw/day. Her stomach, duodenum, jejunum, ileum, caecum and colon were distended with gas. In addition, enlarged adrenal glands were noted and the spleen and thymus were reduced in size. These findings correlated with the observations during the in-life phase, i.e. clinical signs of toxicity and significant lower body weight and food consumption (see previous sections).
Other macroscopic alterations included alopecia and enlarged discolored parathymic lymph nodes, observed for single control females. As this concerned control animals, it was not related to treatment with test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations by treatment up to and including 600 mg/kg bw/day.
All histologic changes were considered to be within the range of background pathology encountered in pregnant non-fasted female rats of this age and strain and for this study type.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations by treatment up to and including 600 mg/kg bw/day.

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

All females were pregnant and had litters with viable fetuses, except for one female at 600 mg/kg bw/day, which was found dead on Day 20 post-coitum and had 13 dead fetuses.
Although the remarkable effects observedone female at 600 mg/kg bw/day, she had 11 viable fetuses.
The numbers of pregnant females, corpora lutea and implantation sites, and pre or postimplantation loss in the control and test groups were similar and in the range of normal biological variation.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Remarks on result:

other:

Remarks:

observation of one spontaneous death in the high dose group with no identified cause of death.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean combined (male and female) fetal body weights were 5.0, 5.2, 5.2 and 4.9 gram for the control, 60, 200 and 600 mg/kg bw/day groups, respectively.
Fetal body weights (both sexes) were slightly lower in the 600 mg/kg bw/day Group. Mean male and female fetal weights were 5.0 (versus 5.2 in the control group) and 4.8 (versus 4.9 in the control group). In addition, the concurrent control values were also relatively low when compared to the available historical control data (mean values: 5.4 gram for males and 5.1 gram for females.
These marginal changes at 600 mg/kg bw/day were caused by one litter, which had a mean fetal body weight of 2.8 gram. The remarkable low fetal body weight of this litter was considered related to the maternal observations during the in-life phase.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to and including 600 mg/kg bw/day.
Mean sex ratios (males:females) were 48:52, 46:54, 49:51 and 51:49 for the control, 60, 200 and 600 mg/kg bw/day groups, respectively.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size of any group.
Mean litter sizes were 11.5, 10.4, 10.9 and 10.8 fetuses/litter for the control, 60, 200 and 600 mg/kg bw/day groups, respectively.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to and including 600 mg/kg bw/day.
External malformations were noticed in one fetus each of the control and high dose Group. The fetus at the 600 mg/kgbw/day group had exencephaly combined with an open eyelid, which were both
seen previously in a historical control fetus. Moreover, this fetus had situs inversus and abnormal lung lobulation as well. At skeletal examination, the exencephaly was substantiated and also two other malformations were revealed, i.e. fused nasal bones and vertebral anomaly with associated rib anomaly. The accumulation of these malformations in one single fetus was considered to have occurred by chance and was not related to treatment.
The control fetus had no lower jaw (although small, fused and malformed mandibles were seen skeletally) and as such was considered a chance finding.
External variations were not seen in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on skeletal morphology following treatment up to and including 600 mg/kg bw/day.
Skeletal malformations were observed in 2 (1), 4 (3), 0 (0) and 3 (3) fetuses (litters) in the 0, 60, 200 and 600 mg/kg bw/day groups, respectively.
In the 600 mg/kg bw/day group, a vertebral centra anomaly and costal cartilage anomaly were noted besides the malformations previously mentioned for one fetus (fused nasal bones and vertebral anomaly with associated rib anomaly). In the 60 mg/kg bw/day group, a vertebral anomaly without associated rib anomaly and three cases of bent limb bones were observed. Two of the control fetuses had bent limb bones as well. The group distribution and or single occurrence of the above mentioned malformations do not suggest a relation to treatment and therefore they were not considered to be toxicologically relevant.
Skeletal variations occurred at an incidence of 82.2%, 94.8%, 65.7% and 79.0% per litter in the 0, 60, 200 and 600 mg/kg bw/day groups, respectively.
Noteworthy is the variation of reduced ossification of the skull for which the incidence in the low dose group (60 mg/kg bw/day) was statistically significantly higher than the control value, causing an increased litter proportion of total skeletal variations for this low dose group as well. Mean litter incidences of this finding were 9.6%, 25.1%, 8.6% and 14.1% per litter in Groups 1, 2, 3 and 4, respectively.
These incidences did not coincide with the fetal weights in the respective dose Groups (5.0, 5.2, 5.2 and 4.9 grams, respectively), Moreover, the other main ossification parameter of unossified metatarsals and metacarpals did not show a dose response as well (mean litter incidences were 17.6%, 18.5%, 11.5% and 16.1% per litter in Groups 0, 60, 200 and 600 mg/kg bw/day, respectively). Therefore, the increased incidence of reduced ossification of the skull in the low dose group was considered to have arisen by chance and not to be toxicologically relevant.
Other skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to and including 600 mg/kg bw/day.
Three viscerally malformed fetuses were observed in this study and all were from the high dose group. This included one fetus of which the malformations were previously mentioned, and two fetuses from the dam with remarkable signs of toxicity, which fetuses either had an aortic arch that was rightsided or interrupted and combined with a ventricular septum defect. Although only cases of situs inversus were seen previously in historical control fetuses, the presence of the two other malformations at the high dose level was not considered treatment related at the single incidences they occurred.
The three visceral variations that were noted in this study all involved the liver (small supernumerary liver lobes, appendix of the liver and discolored liver). As they occurred at low incidences and in the absence of a dose-related incidence, they were not considered to be treatment related.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Accuracy of preparation:

A small response at the retention time of the test item was observed in one of the chromatograms of the Group 1 formulation prepared for use during the treatment period of the dose range finding study. It was considered not to derive from the formulation since the response was not observed in the duplicate study sample. In all other formulations of Group 1, no test item was detected.

The concentrations analysed in the formulations of Group 2, Group 3, Group 4 of the dose range finding and main study and the extra Group of the dose range finding were in agreement with target concentrations (85 -108%,). On one measure day the mean accuracy of the QC samples prepared at the target concentration of 200 mg/g was slightly below 90% (i.e. 85%). This is due to a significant variation within the duplicate. As the mean accuracy was only slightly below 90% and as all test samples and other QC samples were within specification, the analytical method was evaluated as adequate and

the results accepted.

Homogeneity

The formulations of Group 2, Group 4 of the dose range finding and main study and the extra Group of the dose range finding were homogeneous (0.62 to 4.2%).

Stability

Analysis of Group 2, Group 4 and the extra Group of the dose range finding study after storage yielded a relative difference of ≤ 10% (1.9%). Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Results developmental toxicity

Dose level (mg/kg bw/day)	0	60	200	600
Pregnant/total dams	22/22	22/22	22/22	22/22^A
-early resorptions -late resorptions (% per litter)	3.8 0.0	6.3 0.0	5.0 0.0	3.5 0.4
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses	0	0	0	0
Pre-implantation loss (number and percent)	9 (3.2%)	20 (6.7%)	16 (5.7%)	28 (10.5%)
Post-implantation loss (number and percent)	11 (3.8%)	16 (6.3%)	13 (5.0%)	9 (3.8%)
Body weight on day 21	321	318	315	303
Body weight gain day 6-21 (%)	46	43	44	38^**
Gravid uterine weight (g)	76.0	69.8	73.3	70.5
Mean live offspring (number)	11.5	10.4	10.9	10.8
Live offspring (percent)	96.2	93.7	95.0	96.2
Mean fetal body weight males (g)	5.2	5.3	5.3	5.0
Mean fetal body weight females	4.9	5.1	5.0	4.8
Mean fetal body weight (sexes combined)	5.0	5.2	5.2	4.9
Malformations (including runts) number and percent of fetuses per litter	3 (1.8%)	4 (3.9%)	0 (0.0%)	4 (3.0%)
Variations (% per litter) -external -soft tissue -skeletal	0.0 8.8 82.2	0.0 7.4 94.8*	0.0 7.1 65.7	0.0 7.2 79.0

^A: one female died

*P 0.05

^**P 0.1

Applicant's summary and conclusion

Conclusions:

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Accelerator (PT 25E or PT 25E/2) was established at 200 mg/kg bw/day. The offspring and developmental NOAEL was established at 600 mg/kg bw/day, the highest dose tested.

Executive summary:

Mated female Wistar Han rats were assigned to four dose groups, each containing twenty-two animals. The test item was administered once daily by gavage from Day 6 to 20 post-coitum at doses of 60, 200 and 600 mg/kg bw/day. The rats of the control group received the vehicle, propylene glycol, alone.

Accuracy and homogeneity of formulations were demonstrated by analyses.

Maternal findings

One spontaneous death was noted in the 600 mg/kg bw/day group on Day 20 postcoitum. This female showed piloerection and moderate tonic spasms on Day 19 post-coitum. The tonic spasms were observed directly after dosing and recovered after two minutes. Body weights and food consumption were considered to be normal and at necropsy, no macroscopic findings were noted. This female was pregnant and had 13 fetuses, of which no external abnormalities were noted. The reason for this spontaneous death could not be identified, but a relationship to treatment cannot be excluded.

Remarkable clinical signs of toxicity (i.e. rales, piloerection, smacking, lethargy, salivation and hypothermia) in addition to significant body weight loss and reduced food consumption were noted for one single female treated at 600 mg/kg bw/day. Moreover, the gastrointestinal tract was distended with gas, adrenal glands were enlarged and the spleen and thymus were reduced in size. A treatment relation of these observations in a single female is unlikely but cannot be excluded.

Some of the remaining females treated at 600 mg/kg bw/day also showed treatment related and toxicologically relevant effects. Rales was noted for four females, and tremor and lethargy were noted both for three females at 600 mg/kg bw/day. Statistically significantly lower mean body weight gain (incl. corrected for gravid uterus) and food consumption were noted from Day 9 post-coitum onwards, and from Day 6 to Day 15 post-coitum, respectively.

Moreover at 600 mg/kg bw/day, relative liver weights were increased with 13% when compared to controls. As this minimal increase could not be correlated to macroscopic or microscopic findings, this was considered non-adverse.

Treatment at 200 mg/kg bw/day resulted in statistically significantly lower body weight gains on Days 12 and 15 post-coitum. Food consumption was statistically significantly lower from Day 6 to Day 12 post-coitum. As these changes recovered during the remainder of the treatment period, they were considered non-adverse. Relative liver weights were increased with 6%, which was considered non-adverse. Weight gain corrected for gravid uterus was unaffected and no toxicologically relevant clinical signs, macroscopic or microscopic alterations were noted.

No maternal toxicity was observed in the 60 mg/kg bw/day group.

Developmental findings

No developmental toxicity was observed in the 60, 200 and 600 mg/kg bw/day groups.

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for Accelerator (PT 25E or PT 25E/2) was established at 200 mg/kg bw/day. The offspring and developmental NOAEL was established at 600 mg/kg bw/day, the highest dose tested.