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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Remarks:
OECD 407
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-17 - 2012-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented GLP-guideline study
Reason / purpose:
reference to other study
Remarks:
range-finder study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales Des Landes Nordrhein-Westfalen
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS (Wistar (Hsd Cpb:WU))
- Source: Harlan Laboratories BV, Horst, The Netherlands
- Age at delivery: 5-6 weeks
- Weight at study initiation: males: 163-185 g (mean: 174,4 g); females: 135-156 g (mean: 143.3 g)
- Housing: From arrival to tattooing: individually in Makrolon cages Type Ila.
From tattooing to necropsy: in groups with 2 or 3 animals per cage in Makrolon cages Type IV.
Bedding material: Low-dust wood granules (Lignocel BK 8-15; supplier: Ssniff Spezialdiäten Inc. Soest, Westfalen, Germany;
manufacturer: J. Rettenmeier, Ellwangen-Holzmühle, Germany).
Wooden blocks (supplied by Tapvei OY. Kortteinen, Finland) for environmental enrichment will be added to each cage.
As soon as necessary, they will be replaced by new ones.
- Diet (e.g. ad libitum): ad libitum (ssniff R/M-H. 10 mm; Alleinlutter für Ratten und Mäusehaltung: ssniff Spezialdiäten GmbH, Soest, Germany.)
- Water (e.g. ad libitum): ad libitum (Tap water will be given in polycarbonate bottles)
- Acclimation period: approximately one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): Approximately 55 % (Temperature and relative humidity are recorded continuously.)
- Air changes (per hr): > 10 /hour
- Photoperiod (hrs dark / hrs light): 12h/12h (During the light period a radio program is playing.)

IN-LIFE DATES: From: 17 Jan 2012 To: 24 Feb 2012

OTHER:
- identification: Tail tattoos and cards
- Wet cleaning of the floor and tables including disinfection weekly.
Detailed room cleaning and disinfection in intervals as routine.
Continuous pest control using sticky cockroach traps on pheromone basis (purchased from Killgerm GmbH, Neuss, Germany).
- Cages and drinking bottles are exchanged for clean ones routinely. Cage lids and racks are exchanged or cleaned periodically as routine.
Route of administration:
oral: gavage
Vehicle:
other: tap water/Cremophor (98 % / 2 %, v/v)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered orally by gavage in tap water / Cremophor (98% / 2%; v/v). The administration volume was 10 mL/kg body weight. The formulation were prepared as needed and taking into account the analytically determined stability. The formulations were stirred with a magnetic stirrer for several minutes (until an obviously homogenous distribution occurs) before application. They were also stirred during application.
The formulation is stored at room temperature. The formulation is at least stable for 7 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not given
- Amount of vehicle (if gavage): 10 mL / kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the start of treatment, the suitability of the proposed formulations was confirmed by the analyses of concentration and stability. Analyses were carried out under F0012098.
Analyses have shown that in the concentration range from 1 mg/mL to 100 mg/mL the test substance is homogenously distributed in the vehicle and is stable for at least 7 days.
- Concentration/Homogeneity: The homogeneity and the concentrations of the formulations prepared were determined twice during the study.
- Stability: The formulations prepared were analyzed 0, 3, 4, 7 and 8 days after preparation.
Duration of treatment / exposure:
30 days (males) / 31 days (females)
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
40 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
160 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a first pilot study (study No. 18082738) the test item was administered orally by gavage to 3 male and 3 female rats per group, in daily doses of 0, 500 or 1000 mg/kg b.w. per day for an intended period of 2 weeks. Due to severe toxic effects the treatment of the animals of this dose group was stopped on day 4. From day 6 of the study onwards 3 males and 3 females were included in the study and w ere treated with 750 mg/kg for a period of 10 days. Whereas females of the 750 mg/kg group slightly lost body weight after the first treatments and then gained body weight comparable to the control group, males lost body weight after 6 treatments, resulting in 19% lower body weights compared to control at the end of the study. The body weights of animals dosed with 500 mg/kg were unaffected by the treatment. Histopathology revealed in all treatment groups moderate to marked hyperplasia of the forestomach mucosa as well as hyperkeratosis/parakeratosis. Furthermore, erosions of the forestomach mucosa associated with inflammation of the forestomach and in the glandular stomach degeneration/regeneration mainly of surface epithelium and superficial part of the glands were seen.
Based on these results, doses of 0-50-125-250 mg/kg body weight were chosen for a second pilot study. The main findings in this study were changes in the forestomach: a slight hyperplasia of the epithelium was also observed in one animal at 50 mg/kg and all animals at 125 mg/kg and in a more severe grade in all animals at 250 mg/kg. This finding was accompanied by hyperkeratosis in the mid and high dose and a slight inflammation at 250 mg/kg. Based on these results 0, 10, 40, 160 mg/kg were chosen for the present study.

- Rationale for animal assignment (if not random): Appointment of rats to study groups was done by randomization using the procedure within the Pristima System. Rats with extreme body weights were excluded during randomization.
Control and treated animals were handled each in the same manner.

Positive control:
not performed
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Inspections on mortality and morbidity of the animals were performed twice daily. General condition and behavior of animals were checked and recorded daily about 1 hour after administration per gavage.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A careful clinical examination was performed daily prior to treatment. Any clinical signs and abnormalities including changes in skin, fur, eyes, mucous membranes occurrence of secretions and excretions, autonomic activity, changes in gait, posture, and response to handling, clonic/tonic movements, stereotypes and bizarre behavior were recorded as well as time of onset, location and grading. Furthermore, an observation outside the home cage in a standard arena was performed once prior to treatment and weekly thereafter in all animals before treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The individual body weights were determined just before first treatment of animals and daily thereafter. The corresponding application volumes, were filed together with the study raw data and are not reported. Furthermore, body weights were recorded immediately before scheduled necropsies, for calculation of relative organ weights.

FOOD AND WATER CONSUMPTION:
- The food and water consumption per cage were determined weekly. These data were then used to calculate the group means for each period of approximately 7 days. The weight of the food offered at the start of the measurement period minus the weight of the food at the end of the period is defined as the food consumption of the animal in g. Comparable calculation were done for the water intake.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In general, the determinations were performed using standardized procedures subjected to continuous internal and external quality control.
The blood samples for determination of glucose concentrations were taken from one of the caudal veins of non-fasted, non-anesthetized animals.
The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of non-fasted animals anesthetized with C02/room air. The blood obtained was treated as follows:
The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant).
The samples for the determinations of the thromboplastin time (HQUICK) were collected in tubes with sodium-citrate.
The samples for the determinations of electrolyte concentrations remained untreated.
The samples for other biochemical tests were heparinized.
The blood samples for glucose determinations were mixed with perchloric acid ( 1 + 1 0) to precipitate proteins.
- Anaesthetic used for blood collection: No (for determination of glucose), Yes (all other animals)
- Animals fasted: No
- Parameters examined: Leukocytes, erythrocytes, haemoglobin, haematocrit, reticulocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCI 1). mean corpuscular haemoglobin concentration (MCHC), thrombocytes, Hepato Quick, differential blood count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of sceduled necropsy
- Animals fasted: No
- How many animals: all animals of groups 1-4
- Parameters examined: Alanine aminotransferase, aspartate aminotransferase, albumin, protein (total), bile acids, cholesterol, creatinine, urea, glucose, sodium, potassium, total bilirubin.
In general, the determinations were performed using standardized procedures subjected to continuous internal and external quality control.
Occasionally, sample quantity may have been insufficient to permit determination of all intended parameters, or no determination was possible due to technical faults. The planned number of measurements per group may, therefore, not necessarily be obtained in each case.
The blood samples for determination of glucose concentrations were taken from one of the caudal veins of non-fasted, non-anaesthetized animals.
The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of non-fasted animals anesthetized with CO2/room air. The blood obtained was treated as follows:
The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant).
The samples for the determinations of the thromboplastin time (HQUICK) were collected in tubes with sodium-citrate.
The samples for the determinations of electrolyte concentrations remained untreated.
The samples for other biochemical tests were heparinized.
The blood samples for glucose determinations were mixed with perchloric acid (1+10) to precipitate proteins.

OTHER:
Functional Observational Battery (performed once - not blind):
- These examinations were conducted on all animals in dosing week 3/4, earliest about 30 minutes after the treatment.
The following observations/examinations were performed:
- home cage observation: posture, piloerection, gait abnormalities, involuntary motor movements, vocalizations, others.
- observations during handling: ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrimation. nasal discharge, salivation, stains, others.
- open field observations: piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behavior, gait abnormalities, vocalizations, arousal, rearing, defecation, urination.
- reflex / physiological observations: approach response, touch response, auditory response, tail pinch response, pupil size, pupil response (pupil response , righting reflex, grip strength, landing foot splay, body temperature, body weight.

Motor activity:
These examinations were conducted once following the FOB examination.
Motor activity (MA) and locomotor activity (LMA) were examined as activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA). Motor activity was measured as the number of beam interruptions that occurred during the test session.
Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy
All animals living on the date of their scheduled necropsy and all animals to be killed in moribund state were sacrificed by exsanguination under deep isoflurane anaesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination. Changes were described in terms of localization, size, color and consistency whenever appropriate.
If animals died spontaneously during the study they were necropsied at the earliest opportunity. From these animals the organs and tissues were handled as described above.

Organ Weights
From all animals necropsied as scheduled, the organs listed in the following Table 2 marked with "X" in the column "Organ weights" was trimmed and weighed wet, as soon as possible after dissection. The ratio of organ weight to body weight was calculated.

HISTOPATHOLOGY: Yes
Preservation of Tissues
The fixed material was retained (pathology no. 8107). Histological procedures, organs and tissues fixed and evaluated are given in details in the Pathology Report in the Annex (Section 7.8). The scope of organs histopathologically evaluated is shown in Table 2.
Histopathological Technique
Histopathological technique will be performed on tissues marked with "X" in the following Table 2 in the column "Histotechnique up to slides"".
Sections of all tissues will be stained with hematoxylin and eosin (1 l&L ). Cryocuts from liver sections will additionally be stained with Oil Red O.
Osseous tissues will be decalcified before further processing.
Histopathological Evaluation
The tissues in the following Table marked with "X" in the column "Histopathological evaluation" from animals (from decedent animals as far as possible) will be histopathologieally evaluated. If possible treatment-related effects occur this organ will be examined in the lower dose groups. This includes also organs being morphologically connected or fixed with them. The organs of animals of groups 2, 3, which died or were killed prematurely, will be histopathologieally evaluated as described for animals of group 4. If additional organs have to be examined according to these rules, this will be noted in the raw data without amending the study plan..
Statistics:
The statistical evaluation of data related to clinical chemistry, haematology, body and organ weights were performed using routines of the validated Pristima System. Statistical evaluations on FOB, body weight and absolute organ weight data were done using the Dunnett-test. For relative organ weights the Dunnett exact homogenous test after logarithmic transformation were used. Data for food/water intake were statistically evaluated using the adjusted Mann-Whitney U-Test. Clinical pathology data were evaluated using a Dunnett exact homogenous or heterogeneous test, a Dunnett exact homogenous test after logarithmic transformation or the Bonferroni/Mann-Whitney U-test.
All variables that are not dichotomous are described by sex, dose group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum). Statistical tests were not performed for groups, which are smaller than 3.
Statistics of MA/LMA were generated with an evaluation step of the SPADER application. Generation of results was done for duration and interval analysis separately. A preliminary step determined whether there exist significant interactions at all for duration and interval analysis. If so, a Dunnett test based on the GLM Procedure were executed to determine which specific groups differ in a significant way on basis of the computed means with regard to the comparison group.
For statistical evaluations of histopathological data, if any, the PATHDATA program was used and described in detail in the Pathology Report.
Clinical signs:
no effects observed
Description (incidence and severity):
No animal died unscheduled. Therefore, survival was not affected by the treatment with the test substance. At clinical observations no findings were observed.
Mortality:
no mortality observed
Description (incidence):
No animal died unscheduled. Therefore, survival was not affected by the treatment with the test substance. At clinical observations no findings were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight development as well as food and water intake in treated groups was comparable to the respective control group.
Haematological findings:
no effects observed
Description (incidence and severity):
Neither hematology nor clinical chemistry gave evidence for treatment-related effects up to 160 mg/kg.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Neither hematology nor clinical chemistry gave evidence for treatment-related effects up to 160 mg/kg.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Determination revealed slightly increased absolute & relative weights of livers at 160 mg/kg in females & decreased weights of thymus in males of this dose group. The increased mean weight of uterus at 10 mg/kg is mainly caused by a high value of animal27
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings indicating reduced local tolerance were observed at necropsy and at histopathology in the forestomach % small intestine at 160 mg/kg. At necropsy depression/s, thickening or nodule/s of the forestomach mucosa were described.
Details on results:
CLINICAL SIGNS AND MORTALITY
Survival was not affected by the treatment with the test substance. At clinical observations no findings were observed.

BODY WEIGHT AND WEIGHT GAIN
Body weight development as well as food and water intake in treated groups was comparable to the respective control group. Body weight development of males and females was not retarded by the treatment up to 160 mg/kg.

FOOD CONSUMPTION
Mean food intake over the dosing period was comparable to the respective control. Food and water intake in treated groups was comparable to the respective control group.

WATER CONSUMPTION:
The water intake was not relevantly affected by the treatment with the test substance. Food and water intake in treated groups was comparable to the respective control group. The water intake was not relevantly affected by the treatment with the test substance.

FUNCTIONAL OBSERVATIONAL BATTERY:
In week 3/4 a FOB was conducted including home cage observation, observation during handling and in an open field, reflex/physiological observations and measurement of grip strength. The investigation gave no evidence for treatment-related findings.

MOTOR ACTIVITY ASSESSMENT:
Motor activity measurements of male and female rats were made in week 3/4, respectively. The activity determination over the entire 60 minute observation period failed to reveal a significant effect on motor (MA) and locomotor activity (LMA). The results of the 10-minute intervals indicate that there were no relevant differences between treated and control groups In particular, there were no significant differences in the first 10-minute intervals which are regarded as best indicator of increases or decreases in activity before the animals habituate to a minimal level of activity.

HAEMATOLOGY
Hematology gave no evidenced for treatment-related effect on red or white blood or blood coagulation. Neither hematology nor clinical chemistry gave evidence for treatment-related effects up to 160 mg/kg.

CLINICAL CHEMISTRY
Clinical chemistry gave no evidence for treatment-related changes. The only conspicuous findings were decreased activities of ALAT at 10 mg/kg and of ASAT at 160 mg/kg in females. However, due to missing dose dependence, relatively slight differences to control and/or due to the fact that generally increases of these activities indicate adverse effects these changes are not regarded as toxicologically relevant.

GROSS PATHOLOGY / HISTOPATHOLOGY:
At necropsy treatment-related findings were observed in the stomach of males and females treated with 160 mg/kg. One male and 3 females showed depressions of the stomach, in one male it was thickened and in one females a nodule was observed.
Determination of organ weights revealed slightly increased absolute and relative weights of livers at 160 mg/kg in females and decreased weights of thymus in males of this dose group.
The increased mean weight of uterus at 10 mg/kg is mainly caused by the high value of animal No. 27.

In the forestomach, focal or diffuse hyperplasia of the squamous epithelium were observed in most animals dosed at 160 mg/kg. In females, in addition inflammatory infiltrates and edema were seen at a slightly increased incidence at that dose level. One female showed an erosion of the forestomach mucosa. In the duodenum or jejunum, increased goblet cells were seen in the small intestine at 160 mg/kg. No signs of inflammation were present in the small intestine. In one female at 40 mg/kg, an inflammatory infiltrate and minimal edema were observed at the limiting ridge between forestomach and glandular stomach. This lesion is likely of spontaneous origin since it was not associated with any changes of the forestomach epithelium.
There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg.
In conclusion, treatment-related findings indicating reduced local tolerance were observed at necropsy and at histopathologically in the forestomach and small intestine at 160 mg/kg. At necropsy depression/s, thickening or nodule/s of the forestomach mucosa were described. These findings correlated with diffuse hyperkeratosis, focal hyperplasia and erosion of the forestomach mucosa. Inflammatory infiltrates and edema were seen at slightly increased incidences at 160 mg/kg. In the duodenum and jejunum, increased goblet cells were seen in the mucosa at 160 mg/kg. Furthermore, in the duodenum or jejunum, increased goblet cells were seen in the small intestine. This indicates increased mucous production related to decreased local tolerance.
There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg.

Daily oral treatment of rats with the test item at doses of 0, 10, 40, 160 mg/kg b.w. for a period of 4 weeks resulted in local effects in the forestomach and small intestine.
Under the conditions described the no observed adverse effect level (NOAEL) for administration of the test substance to male and female Wistar rats was 40 mg/kg bw.
This NOAEL is set due to local irritating effects. There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local irritation in forestomach
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg.
Remarks on result:
other: highest dose tested
Critical effects observed:
not specified

Table‎ 3: Body Weights (g)

Sex

Males

Females

Dose

 (mg/kg)

0

10

40

160

0

10

40

160

Day

 

 

 

 

 

 

 

 

1

174.0

176.0

175.2

172.2

142.2

143.0

143.6

144.2

2

180.0

179.4

182.4

178.2

144.8

148.6

146.4

146.2

3

189.0

188.6

190.4

184.6

146.8

149.2

150.2

149.8

4

196.4

195.2

197.2

191.4

151.2

152.6

153.6

154.0

5

203.2

201.0

205.4

200.2

154.8

155.8

157.6

159.2

6

211.6

210.0

213.0

207.2

156.6

161.8

160.8

164.0

7

218.0

215.0

219.8

214.8

158.0

163.0

163.2

164.8

8

224.8

221.8

226.8

222.0

162.2

167.0

169.2

169.0

9

232.2

229.4

234.6

229.2

168.4

170.6

172.4

175.6

10

238.2

235.6

241.2

236.8

169.4

173.6

177.0

178.4

11

247.0

242.6

248.6

242.6

172.0

176.0

175.8

177.8

12

250.2

248.8

253.0

248.8

176.2

180.8

182.6

183.6

13

257.6

252.2

259.2

254.4

180.0

184.2

185.2

187.6

14

266.6

260.0

268.0

263.8

181.6

187.8

189.6

192.2

15

269.8

264.8

273.2

267.4

184.0

187.4

188.6

190.8

16

278.2

270.8

280.6

274.4

189.4

190.6

195.2

195.2

17

279.4

274.2

284.4

277.2

192.4

194.6

197.0

199.8

18

287.4

279.0

290.8

282.4

193.0

198.4

199.4

203.0

19

289.4

285.0

293.8

283.8

194.6

195.0

199.8

200.6

20

295.6

288.2

299.6

289.8

198.4

199.2

204.8

205.2

21

299.2

294.6

304.8

295.6

203.2

203.2

205.4

211.4

22

303.6

295.4

307.6

297.2

202.0

208.0

209.2

212.8

23

306.2

300.2

311.0

302.0

202.4

205.4

207.4

210.6

24

311.2

304.0

316.8

308.2

206.0

205.6

212.6

214.6

25

314.8

306.6

320.0

310.4

209.6

210.2

214.8

217.6

26

317.8

312.8

324.2

313.2

210.2

213.8

216.0

220.4

27

323.6

314.8

329.4

316.6

211.8

214.8

217.4

219.2

28

326.6

318.0

332.2

321.8

213.0

214.8

221.4

221.8

29

331.2

324.2

335.4

326.4

218.4

218.8

222.2

226.4

30

336.6

328.8

344.0

331.8

217.4

223.8

224.8

228.0

31

 

 

 

 

219.0

221.6

223.8

224.0

Table 4: -Mean Daily Food Intake

Group means

Dose

Days

g/animal

g/kg body weight

ppm

 

per day

per day

Male

0

1-29

25.0

89.9

10

1-29

23.9

87.8

40

1-29

25.0

88.8

160

1-29

24.8

90.5

Female

0

1-29

17.4

91.4

10

1-29

18.0

93.2

40

1-29

17.5

89.5

160

1-29

18.8

95.2

Table5: -Mean Daily Water Intake

Group means

Dose

Days

g/animal

g/kg body weight

ppm

 

per day

per day

Male

0

1-29

28.8

103.8

10

1-29

30.9

113.5

40

1-29

31.4

112.7

160

1-29

30.9

111.4

Female

0

1-29

21.1

116.7.

10

1-29

22.1

114.3

40

1-29

21.4

109.4

160

1-29

22.6

114.2

Table 6: Haematology

Dose

ERY

HB

HCT

MCH

MCHC

MCV

RETI

THRO

Hep-Quick

(mg/kg)

T/l

g/l

l/l

pg

g/l Ery

fl

%

G/l

sec.

Males - Day 31

0

8.398

156.0

0.5218

18.58

299.0

62.14

2.10

1075.0

34.18

10

8.484

158.0

0.5328

18.60

296.2

62.84

1.90

 981.2

36.34

40

8.396

157.6

0.5300

18.80

297.2

63.22

2.08

 978.2

34.16

160

8.426

156.0

0.5260

18.54

297.0

62.44

2.42

1058.8

33.34

Female - Day 32

0

8.276

150.4

0.5126

18.20

293.8

61.94

1.84

1150.8

32.10

10

8.470

155.0

0.5350

18.30

289.8

63.18

2.12

 975.4*

33.50

40

8.368

151.4

0.5112

18.10

296.2

61.12

1.74

1119.4

32.00

160

8.534

154.6

0.5234

18.12

295.4

61.34

1.82

1100.2

32.20

 

Table 7: Differential Blood Count

Dose

LEUCO

LYM

NEUTRO

Basophils

EOS

MONO

Atypical

(mg/kg)

G/l

G/l

G/l

G/l

G/l

G/l

G/l

Males - Day 31

0

12.126

10.742

0.828

0.100

0.100

0.272

0.082

10

10.948

 9.740

0.738

0.072

0.112

0.198

0.088

40

11.066

 9.852

0.706

0.070

0.106

0.234

0.100

160

10.950

 9.622

0.844

0.072

0.102

0.218

0.090

Females - Day 32

0

 9.112

 8.074

0.608

0.070

0.086

0.194

0.076

10

 8.362

 7.426

0.580

0.056

0.076

0.154

0.076

40

 8.966

 7.854

0.700

0.068

0.106

0.160

0.072

160

 8.636

 7.596

0.650

0.074

0.074

0.174

0.078

Table 8: Clinical Chemistry:

Dose

ALAT

ASAT

(mg/kg)

U/l

U/l

Males Day 31

0

55.72

60.58

10

61.16

63.44

40

59.58

58.64

160

61.66

60.54

Females Day 32

0

55.88

64.22

10

45.82**

57.68

40

51.12

54.32

160

48.68

56.92**

**   = significantly different at p <= 0.01

Table 9: Clinical Chemistry: Substrates

Dose

GLUCOSE

CHOL

CREA

UREA

Bili-t

S-Bile ac.

Protein

Albumin

(mg/kg)

mmol/l

mmol/l

µmol/l

mmol/l

µmol/l

µmol/l

g/l

g/l

Males - Day 31 

0

5.620

2.412

51.2

7.436

0.10

23.52

67.24

36.44

10

5.294

2.056

54.4

6.878

0.02

27.64

67.68

36.86

40

5.454

2.080

51.0

6.640

0.00

22.92

66.32

36.44

160

5.334

2.364

51.0

7.478

0.06

31.02

65.78

36.14

Females - Day 32

0

5.148

1.728

54.8

7.184

0.06

21.68

65.84

37.54

10

4.978

1.974

53.8

7.032

0.04

27.76

64.84

36.50

40

5.124

2.140

53.2

6.790

0.02

13.66

65.80

37.14

160

5.192

2.100

52.6

8.074

0.00

10.16

66.90

37.36

  

Table 10: Clinical Chemistry: Electrolytes

 

Na

K

Dose

mmol/l

mmol/l

Males

Day 31

 

0 mg/kg

147.6

6.28

10 mg/kg

147.2

6.12

40 mg/kg

147.6

6.10

160 mg/kg

147.4

6.04

Females

Day 32

 

0 mg/kg

145.6

5.44

10 mg/kg

145.6

5.34

40 mg/kg

146.0

5.42

160 mg/kg

146.4

5.14

Conclusions:
The GLP-study was performed according to the OECD Guideline 407 in rats without deviations and is considered to be reliable without restrictions (reliability Klimisch 1). The test material did induce a response and the results show that the test item has a NOAEL of 40 mg/kg bw.
Executive summary:

In a repeated dose study (Schadt, 2012, according to OECD 407), the substance was administered orally by gavage to 5 male and 5 female Wistar (Hsd Cpb:WU) rats per dose group using tap water / Cremophor (98% / 2%; v/v)as vehicle, in daily doses of 0, 10, 40, 160 mg/kg bw for a period of 4 weeks. The animals were regularly observed and weighed and feed and water intakes were determined. In addition, clinical laboratory investigation of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation.

Survival was not affected by the treatment with the test substance. At clinical observations no findings were observed. Body weight development as well as food and water intake in treated groups was comparable to the respective control group. Neither haematology nor clinical chemistry gave evidence for treatment-related effects up to 160 mg/kg. Treatment-related findings indicating reduced local tolerance were observed at necropsy and at histopathologically in the forestomach and small intestine at 160 mg/kg. At necropsy depression/s, thickening or nodule/s of the forestomach mucosa were described. These findings correlated with diffuse hyperkeratosis, focal hyperplasia and erosion of the forestomach mucosa. Inflammatory infiltrates and oedema were seen at slightly increased incidences at 160 mg/kg. One female showed an erosion of the forestomach mucosa. The forestomach is particularly sensitive to local irrtancy since the test compound remains there for prolonged time period after oral uptake. These findings are an indication of reduced local tolerance. Furthermore, in the duodenum and jejunum, increased goblet cells were seen in the mucosa at 160 mg/kg. Furthermore, in the duodenum or jejunum, increased goblet cells were seen in the small intestine. This indicates increased mucous production related to local irritation.

In conclusion, daily oral treatment of rats with the test item resulted in local effects in the forestomach and small intestine. Under the conditions described the no observed adverse effect level (NOAEL) for administration of 1,1'dithiobis[hexahydro-2H-azepin-2 -one to male and female Wistar rats was 40 mg/kg bw. This NOAEL is set due to local irritating effects. There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg. Therefore, the NOAEL based on systemic effects is >160 mg/kg bw/day.

Reason / purpose:
reference to other study
Remarks:
Range-finding study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1 February 2017 - 5 March 2017 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Range-finding study for OECD 414
Reason / purpose:
reference to other study
Remarks:
OECD 407
Reason / purpose:
reference to other study
Remarks:
OECD 414
Qualifier:
according to
Guideline:
other: Range-finding study for OECD 414
Principles of method if other than guideline:
- Principle of test: Range-finding study for OECD 414 under similar conditions
- Short description of test conditions: Three groups of six females received Caprolactam disulfide at doses of 10, 40 or 160 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating.
- Parameters analysed / observed: Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy.
GLP compliance:
no
Remarks:
study was conducted in a facility which operates in accordance with Good Laboratory Practice principles; however no claim of GLP compliance was intended nor is made for this study
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15-25°C)
- Stability under test conditions: Stability of the formulation was confirmed for up to 8 days when stored refrigerated (2 to 8°C), and 24 hours when stored at ambient temperature (15 to 25°C)) at 1 and 100 mg/ml.
Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 81 to 87 days old
- Weight at study initiation: 185 to 227 g
- Fasting period before study: no
- Housing:
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
The cages constituting each group were blocked by group and mounted in batteries.
Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Environmental Enrichment:
Aspen chew block : A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.
Number of animals per cage:
Acclimatization: up to four animals
During pairing: one (stock) male and one female
Gestation: one female
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water ad libitum from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Nine days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC.
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark

IN-LIFE DATES: From: 1 February 2017 To: 3 to 5 March 2017
Route of administration:
oral: gavage
Vehicle:
other: 98% Water with 2% Kolliphor EL(cremophor) v/v
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Comparability with previous studies
- Concentration in vehicle: 0, 1, 4, 16 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Stability was confirmed for up to 8 days when stored refrigerated (2 to 8°C), and 24 hours when stored at ambient temperature (15 to 25°C)) at 1 and 100 mg/ml.

CHROMATOGRAPHIC CONDITIONSCHROMATOGRAPHIC CONDITIONS
High performance liquid chromatograph (HPLC)
Column: Poroshell 120 SB-C18 2.7 µm, 150 × 4.6mm
Column temperature: 45ºC
Sample temperature: Ambient
Mobile phase: Acetonitrile/0.05% aqueous formic acid 50/50 v/v
Flow rate: 0.8 mL/min
Rinse solvent: Acetonitrile/water 50/50 v/v
Detector wavelength: UV, 252 nm
Injection volume: 10 µL
Time constant: 1.0 sec
Sampling rate: 2 points/sec
Run time: 8 minutes

These conditions were established using a Waters Alliance 2695 separation module and 2487 detector.
Calibration standard range: 2 µg/mL – 10 µg/mL.
Extraction solvent: Acetonitrile / 0.05% aqueous formic acid 80/20 v/v
Diluent: Acetonitrile / 0.05% aqueous formic acid 50/50 v/v
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Length of cohabitation: until pregnancy
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. When positive evidence of mating was detected, this day referred to as day 0 of pregnancy.
Duration of treatment / exposure:
Day 6-19 of pregnancy
Frequency of treatment:
daily
Duration of test:
mating to necropsy (day 20 of pregnancy), 22-24 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
160 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 females, 6 stock males (not part of the study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses used in this study (0, 10, 40 and 160 mg/kg/day) were selected in conjunction with the Sponsor.
Based on data obtained on a previous study conducted by the Sponsor (OECD 407) Caprolactam disulfide was administered daily by oral gavage to male and female Han Wistar rats for four weeks at dose levels of 0, 10, 40 and 160 mg/kg/day. There was no evidence of any systemic effects related to dosing with test compound up to and including 160 mg/kg/day. There were no deaths and no effects upon clinical signs, body weight, food consumption, hematology and blood chemistry.
Treatment-related findings indicating reduced local tolerance were observed at necropsy and at histopathologically in the forestomach and small intestine at 160 mg/kg. As the present preliminary study was for duration of two weeks of treatment, and there were no deaths or adverse effects on clinical condition, body weight or food consumption, a dose level of 160 mg/kg/day was selected as the high dose level on this study. Low and intermediate dose levels of 10 and 40 mg/kg/day were selected to give an approximate 4-fold difference between dose levels.
The choice of the same dose levels as were tested on the four week toxicity study permitted a robust evaluation of the sensitivity of the pregnant versus non-pregnant female.
- Rationale for animal assignment (if not random): random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing: Detailed observations were recorded daily at the following times in relation to dose administration: Pre-dose observation / One to two hours after completion of dosing / As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION: Yes
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

OTHER: Reproductive assessment
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, including cervix and ovaries
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of Fetuses (live and dead)
Fetal examinations:
All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus was recorded. Grossly normal fetuses were discarded.
Statistics:
No statistical evaluation was performed on this study.
Indices:
Pre-implantation loss, Post-implantation loss
Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths, clinical signs or signs observed following dose administration.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no deaths, clinical signs or signs observed following dose administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean bodyweight and bodyweight gain for females receiving 10, 40 or 160 mg/kg/day was similar to that of the Controls and were considered to be unaffected by treatment with Caprolactam disulfide.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was considered unaffected by treatment with Caprolactam disulfide during Days 6- 19 of gestation when compared with Controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females treated with Caprolactam disulfide up to dose levels of 160 mg/kg/day on Day 20 of gestation did not reveal any findings considered to be related to treatment.
Macroscopic examination of the fetuses did not reveal any findings that could be related to maternal treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Gravid uterine weight was essentially similar across all groups of females, with only females that received 160 mg/kg/day having a slightly lower adjusted maternal bodyweight change (Day 6-20) when compared with Controls (approximately 64% of Controls): this was due to 2/6 females with a very low adjusted weight gain (3-4 g compared with the lowest Control gain of 13 g).
Number of abortions:
no effects observed
Description (incidence and severity):
All females were pregnant. The following assessment is based on the 6, 6, 6 and 6 females with live young at termination on Day 20 of gestation in the Control group and at 10, 40 and 160 mg/kg/day, respectively.
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of corpora lutea, implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of corpora lutea, implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Post implantation loss was higher for females receiving 160 mg/kg/day when compared with Controls, this was considered due to one female (4F 21) having a high post implantation loss of 30.8% which is atypical for this group and not considered related to treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of corpora lutea, implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Early or late resorptions:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of corpora lutea, implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of corpora lutea, implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of corpora lutea, implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Pregnancy was terminated on gestation day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females were pregnant.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 160 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in number of pregnant
necropsy findings
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Placental, litter and fetal weights were unaffected by treatment with Caprolactam disulfide when compared with Controls.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Placental, litter and fetal weights were unaffected by treatment with Caprolactam disulfide when compared with Controls.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Placental, litter and fetal weights were unaffected by treatment with Caprolactam disulfide when compared with Controls.
Changes in postnatal survival:
not examined
Description (incidence and severity):
Pregnancy was terminated on gestation day 20.
External malformations:
no effects observed
Description (incidence and severity):
External examination of the fetuses did not reveal any abnormalities related to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
external malformations
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
External examination of the fetuses did not reveal any abnormalities related to treatment at the highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

see attachments

Conclusions:
The study was perfomed as a range-finding study for a OECD TG 414 study was sufficiently documented, hence, the results can be considered sufficiently reliable to assess partly the developmental toxicity of Caprolactam disulfide in rats and to select a dose level for the main study. No effects related to treatment were observed up to the highest dose tested, 160 mg/kg bw/d, but doses were considered to be well-chosen as other repeated dose toxicity studies revealed i.a. a NOAEL of 40 mg/kg bw/d for general toxicity.
It was concluded from this study that the dosage of 160 mg/kg/day that dose levels of 10, 40 and 160 mg/kg/day or slightly higher would be suitable for the main embryo-fetal toxicity study, and that 160 mg/kg/day was the maternal no observed-adverse-effect-level (NOAEL) and 160 mg/kg/day was the no observed adverse-effect-level (NOAEL) for embryo-fetal survival and development.
Executive summary:

The purpose of this range-finding study was an assessment of the influence of Caprolactam disulfide on embryo-fetal survival and development in the Han Wistar rat and to establish suitable doses for a main embryo-fetal toxicity study.

Three groups of six females received Caprolactam disulfide at doses of 10, 40 or 160 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 98% water with 2% Kolliphor EL (cremophor) v/v at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy.

Results

There were no deaths, and no clinical signs or signs observed following dose administration.

Bodyweight gains of the females receiving 10, 40 or 160 mg/kg/day were similar to Control values, and there was no adverse effect of treatment on the gravid uterus weight but maternal weight gain adjusted for the uterine weight was slightly low at 160 mg/kg/day. Food consumption was not affected as a result of treatment with Caprolactam disulfide.

There were no observations at necropsy that were attributable to treatment. All animals were pregnant. Caprolactam disulfide did not have any adverse effect on implantations and pre or post implantation loss, sex ratio or live young in females receiving up to 160 mg/kg/day. There was no effect of treatment on placental or fetal weight in animals receiving up to 160 mg/kg/day.

External examination of the fetuses did not reveal any abnormalities related to treatment.

Conclusion

Oral administration of Caprolactam disulfide to Han Wistar rats during Days 6 to 19 of gestation at a dose of 10, 40 or 160 mg/kg/day was well tolerated and did not result in any deaths, signs of toxicity or any adverse effects on embryo-fetal survival and development.

Dose levels of 0, 10, 40 and 160 mg/kg/day were well tolerated with no clinical signs and no effect upon body weight or food consumption but adjusted body weight gain was slightly low at 160 mg/kg/day. There was no clear effect of maternal treatment upon numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss. Placental, litter and fetal weights were also all unaffected by treatment.

Based on these results it was concluded that dose levels of 10, 40 and 160 mg/kg/day or slightly higher would be suitable for the main embryo-fetal toxicity study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
current guideline
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
current guideline
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
current guideline
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Health of the Government of the United Kingdom
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15-25°C)
- Stability under test conditions: Stability of the formulation was confirmed for up to 8 days when stored refrigerated (2 to 8°C), and 24 hours when stored at ambient temperature (15 to 25°C)) at 1 and 100 mg/ml.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 77 to 83 days old
- Weight at study initiation: 177 to 211 g (Day 0 of gestation)
- Fasting period before study: no
- Housing:
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
The cages constituting each group were blocked by group and mounted in batteries.
Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Environmental Enrichment:
Aspen chew block : A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.
Number of animals per cage:
Acclimatization: up to four animals
During pairing: one (stock) male and one female
Gestation: one female
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water ad libitum from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Five days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC.
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes (per hr): not stated, filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark

IN-LIFE DATES: From: 8 March 2017 (animal arrival) To: 3 to 6 April 2017 (necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 98% Water with 2% Kolliphor EL(cremophor) v/v
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Comparability with previous studies
- Concentration in vehicle: 0, 2.5, 7.5, 25 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Stability was confirmed for up to 8 days when stored refrigerated (2 to 8°C), and 24 hours when stored at ambient temperature (15 to 25°C)) at 1 and 100 mg/ml.

CHROMATOGRAPHIC CONDITIONSCHROMATOGRAPHIC CONDITIONS
High performance liquid chromatograph (HPLC)
Column: Poroshell 120 SB-C18 2.7 µm, 150 × 4.6mm
Column temperature: 45ºC
Sample temperature: Ambient
Mobile phase: Acetonitrile/0.05% aqueous formic acid 50/50 v/v
Flow rate: 0.8 mL/min
Rinse solvent: Acetonitrile/water 50/50 v/v
Detector wavelength: UV, 252 nm
Injection volume: 10 µL
Time constant: 1.0 sec
Sampling rate: 2 points/sec
Run time: 8 minutes

These conditions were established using a Waters Alliance 2695 separation module and 2487 detector.
Calibration standard range: 2 µg/mL – 10 µg/mL.
Extraction solvent: Acetonitrile / 0.05% aqueous formic acid 80/20 v/v
Diluent: Acetonitrile / 0.05% aqueous formic acid 50/50 v/v
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Length of cohabitation: until pregnancy
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. When positive evidence of mating was detected, this day referred to as day 0 of pregnancy.
Duration of treatment / exposure:
Day 6-19 of pregnancy
Frequency of treatment:
daily
Duration of test:
mating to necropsy (day 20 of pregnancy), 22-25 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20 females, 20 stock males (not part of the study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study (0, 25, 75 and 250 mg/kg/day) were selected in conjunction with the Sponsor.
The dose levels were chosen based on the results of the preliminary study for effects on embryo-fetal development in the Han Wistar rat by oral gavage administration.
In that study, dose levels of 10, 40 and 160 mg/kg/day were well tolerated with no clinical signs and no effect upon body weight or food consumption. There was no clear effect of maternal treatment upon numbers of corpora lutea, implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss. Placental, litter and fetal weights and litter size were also all unaffected by treatment.
As there were no clear treatment related findings, a dose level of 250 mg/kg/day was selected for the high dose (250 mg/kg/day was assessed on a 14 day dose range finder study where the main findings were changes in the forestomach, but not deaths). This was to try and elicit a slight treatment related response, as required by the regulatory authorities.
Therefore it was considered that dose levels of 25, 75 and 250 mg/kg/day would be suitable for use on this main study.
- Rationale for animal assignment (if not random): random
- Other: The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing: Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration: One to two hours after completion of dosing, As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

OTHER: Reproductive Assessment
The following were recorded for all animals:
Uterus: Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn, number of Corpora lutea, Implantation sites, Resorption sites (classified as early or late), Fetuses (live and dead).
Apparently non pregnant animals: The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski, E, 1964)].
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, including cervix and ovaries
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses (live and dead)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter; 50% Sexed internally and eviscerated
- Skeletal examinations: Yes
- Head examinations: Yes:
Statistics:
Due to limitations of this free text field, see "Any other information on materials and methods"
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) x 100 / Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) x 100 / Number of implantations

All group values and SD (as appropriate) were calculated from the individual litter values.
Historical control data:
available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or signs observed following dose administration that were considered to be related to treatment with Caprolactam disulfide.
Two females receiving 250 mg/kg/day were observed with slight piloerection on Day 19 of gestation.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall group mean bodyweight gain (Days 6-20) and bodyweight gain on Days 6-7, 12-13 and 15-16 of gestation was slightly but statistically significantly lower for females receiving 250 mg/kg/day when compared with Controls.
Mean bodyweight and bodyweight gain for females receiving 25 or 75 mg/kg/day was similar to that of the Controls and were considered to be unaffected by treatment with Caprolactam disulfide.
Gravid uterine weight was essentially similar across all groups of females, with only females that received 250 mg/kg/day having a slightly but statistically significant lower adjusted maternal bodyweight change (Day 6 20) when compared with Controls (approximately 83% of Controls).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Overall food consumption was considered unaffected by treatment with Caprolactam disulfide during Days 6- 19 of gestation when compared with Controls.
Food intake was slightly but statistically significantly lower during Days 6-9 of gestation for females receiving 250 mg/kg/day when compared with Controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females treated with Caprolactam disulfide up to dose levels of 250 mg/kg/day on Day 20 of gestation did not reveal any findings considered to be related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Sex ratio (% male) and pre-implantation loss was statistically significantly higher for females that received 25 mg/kg/day when compared with Controls, however, as the mean percentage of pre implantation loss was within the Historical Control Data range (4.6-11.4%, 4 main embryo-fetal studies, 2016-2017) and there was no dose response, hence these findings were not considered related to treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Early or late resorptions:
no effects observed
Description (incidence and severity):
For females examined on Day 20 of gestation litter data showed no clear effects of maternal treatment, with mean numbers of implantations, early, late and total resorptions, number of live young and sex ratio and the extent of pre and post-implantation loss, essentially similar to Controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
none stated
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Pregancy was terminated on Day 20.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Pregancy was terminated on Day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female receiving 250 mg/kg/day was not pregnant, all other females were pregnant.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in number of pregnant
necropsy findings
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Placental, litter and fetal weights were unaffected by treatment with Caprolactam disulfide when compared with Controls.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
number of live young essentially similar to Controls
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio (% male) and pre-implantation loss was statistically significantly higher for females that received 25 mg/kg/day when compared with Controls, however, as the mean percentage of pre implantation loss was within the Historical Control Data range (4.6-11.4%, 4 main embryo-fetal studies, 2016-2017) and there was no dose response, hence these findings were not considered related to treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Placental, litter and fetal weights were unaffected by treatment with Caprolactam disulfide when compared with Controls.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
In all groups that received Caprolactam disulfide, there was a slight increased incidence of delayed ossification of cranial centres when compared to Controls, but this incidence was within the Historical Control Data range. A delay in ossification is a transient stage in fetal development and not considered adverse.
At 25 mg/kg/day there was an isolated high incidence of 20 thoracolumbar vertebrae compared to Control. This finding was outside of the Historical Control Data range, but in the absence of any other vertebral configuration abnormalities, this finding is not thought to be adverse or related to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
highest dose tested

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: vertebra
Description (incidence and severity):
At 25 mg/kg/day there was an isolated high incidence of 20 thoracolumbar vertebrae compared to Control. This finding was outside of the Historical Control Data range, but in the absence of any other vertebral configuration abnormalities, this finding is not thought to be adverse or related to treatment.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

see attachments

Applicant's summary and conclusion

Conclusions:
The study was perfomed acc. OECD TG 414 under GLP and was sufficiently documented, hence, the results can be considered sufficiently reliable to assess the developmental toxicity of Caprolactam disulfide in rats. No effects related to treatment were observed up to the highest dose tested, 250 mg/kg bw/d, but doses were considered to be well-chosen as other repeated dose toxicity studies revealed i.a. a NOAEL of 40 mg/kg bw/d for general toxicity.
It was concluded from this study that the dosage of 250 mg/kg/day was the maternal no observed-adverse-effect-level (NOAEL) and 250 mg/kg/day was the no observed adverse-effect-level (NOAEL) for embryo-fetal survival and development.
Executive summary:

The purpose of this study acc. OECD TG 414 under GLP was an assessment of the influence of Caprolactam disulfide on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat.

Three groups of 20 females received Caprolactam disulfide at doses of 25, 75 or 250 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 98% water with 2% Kolliphor EL (cremophor) v/v at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. 

Results

Maternal clinical condition, bodyweight, food consumption and macroscopic evaluation were not adversely affected by treatment with Caprolactam disulfide at doses up to 250 mg/kg/day when compared with Control animals. At 250 mg/kg/day, there was a slight reduction in mean body weight gain during Days 6-7 of gestation and marginal reductions in adjusted body weight gain, and mean food consumption during Days 6-9 of gestation.

Embryo fetal survival, growth and development were unaffected by treatment with Caprolactam disulfide at doses up to 250 mg/kg/day.

Conclusion

It was concluded from this study that the dosage of 250 mg/kg/day was the maternal no‑observed-adverse-effect-level (NOAEL) and 250 mg/kg/day was the no‑observed‑adverse-effect-level (NOAEL) for embryo-fetal survival and development.