Reaction mass of L-xylo-hex-2-ulosonic acid and ascorbic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2016 - 27 October 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:

Reaction mass of L-xylo-hex-2-ulosonic acid and ascorbic acid

EC Number:

932-019-3

Molecular formula:

not applicable to reaction mass

IUPAC Name:

Reaction mass of L-xylo-hex-2-ulosonic acid and ascorbic acid

Test material form:

liquid

Details on test material:

- State of aggregation: brown liquid
- Density: 1.29 at 20 °C

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a freezer (≤ -15°C) under nitrogen
- Solubility and stability of the test substance in the solvent/vehicle: stability for at least 3 hours at room temperature is confirmed over the concentration range 1 to 200 mg/mL in water

OTHER SPECIFICS: A correction was made for the purity/composition of the test item. The purity of the test item was 22%, therefore a correction factor of 0.22 was used.

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: 173-246 g
- Fasting period before study: none
- Housing: individually housed in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days prior to treatment

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: 29 July 2016 (first delivery of mated females) To: 27 October 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 3 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test item (1.29). A correction was made for the purity/composition of the test item. A correction factor of 0.22 was used.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on two days during the treatment phase (11 and 21 October 2016), according to a validated method. 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%.
See section "Any other information on results, including tables" for the results of analytical determination.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant. Untreated females were time-mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the test facility.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive

Frequency of treatment:

Daily

Duration of test:

14 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Concurrent vehicle controls

Doses / concentrations 2

Dose / conc.:

88 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

264 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

880 mg/kg bw/day

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 the results of the dose range finding study.
Treatment at 1000 mg/kg resulted in one female sacrificed in extremis on Day 20 post-coitum as she showed gasping, lethargy and piloerection. At necropsy, dark red foci and an irregular surface of the glandular mucosa were noted and the stomach was distended with gas. In addition, two other females at 1000 mg/kg showed rales and one female showed lethargy on Days 14-15 post-coitum.
Body weight gains and food consumption of one of the two females showing clinical signs, and one other female were significantly lower compared to controls.
The other females at 1000 mg/kg had normal body weights and food consumption. Macroscopic examination revealed no toxicologically relevant findings. No maternal toxicity was observed at 100 and 300 mg/kg and no developmental toxicity was observed by treatment up to 1000 mg/kg.
All females were found to be pregnant with viable fetuses. No treatment related changes for the number of post-implantation loss were observed.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily for clinical signs 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
- Time schedule for examinations: 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: ovaries, uterine horn

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:

The following indices were calculated:
Pre-implantation loss (%) = ((number of corpora lutea - number of implantation sites)/number of corpora lutea) x 100%
Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/number of implantation sites) x 100%
Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter)/(number of viable fetuses/litter)) x 100%

Historical control data:

Historical control data are available from the same test facility for the period of 2014-2015 and are reported in section "Other remarks on results including tables".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For clinical signs on 6 unscheduled deaths in the high-dose group see section on mortality.
Clinical signs of toxicity were also noted for twelve of the remaining females at 880 mg/kg bw/day. These consisted of hunched posture, rales, piloerection, quick breathing, lethargy, decreased locomotor activity, squeaking and/or chromodacryorrhoea. In addition, regurgitation of the test item via the nose was observed directly after dosing of one female on one single day (Day 12 post-coitum). Only 4/22 high dose females showed no toxicologically relevant clinical signs.
Salivation seen after dosing for all females at 880 mg/kg bw/day (including the unscheduled deaths described in the section on mortality) was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
No clinical signs of toxicological relevance were noted at 88 and 264 mg/kg bw/day. Incidental findings that were noted included alopecia and a nodule of the left eyelid. 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.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment at the highest dose level of 880 mg/kg bw/day resulted in six unscheduled deaths, of which a relationship to treatment with test item cannot be excluded.
One high dose female was found dead on Day 13 post-coitum. Toxicologically relevant clinical signs observed on Day 12 post-coitum included lethargy, hunched posture and piloerection. Significant body weight loss of 18% was noted between Days 9-12 post-coitum and her food consumption was significantly reduced to 5 gram/day (compared to a mean control value of 21 gram/day) on these days. Except for emaciation and advanced autolysis, no macroscopic alterations were observed that could explain the moribund condition of this female.
In addition, five females of the highest dose group were sacrificed in extremis between Days 10-17 post-coitum due to humane endpoints.
In the first female in the afternoon of Day 9 post-coitum, gasping and shallow respiration in combination with squeaking were observed. Moreover, rales, hunched posture, piloerection and labored respiration in combination with squeaking were observed in the morning of Day 10 post-coitum (i.e. before dosing). Based on these findings, it was decided to sacrifice this female on Day 10 post-coitum. On Days 11 and 12 post-coitum, toxicologically relevant clinical signs were noted for two other females, consisting of lethargy, hunched posture, quick or slow breathing, labored and deep respiration, rales, piloerection, a pale appearance, squeaking and/or a red snout. In combination with significant body weight loss of respectively 18% and 13% over Days 9-12 post-coitum and remarkable low food intake on these days, it was decided to sacrifice these two females on Day 12 post-coitum before dosing. On Day 14 post-coitum, another female was sacrificed before dosing as she showed lethargy, hunched posture, rales, piloerection, squeaking and a red snout. The last unscheduled sacrifice occurred in the afternoon of Day 17 post-coitum (i.e. after dosing), as gasping was observed.
No mortality occurred in the other groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gains were lower at 880 mg/kg bw/day from Day 9 post-coitum onwards when compared to controls. This was statistically significant for body weight gains on Days 9 to 15 post-coitum.
No toxicologically relevant changes in body weight and body weight gain were noted at 88 and 264 mg/kg bw/day.
Mean corrected body weight gain for gravid uterus were 31, 26, 29 and 27 gram for the control, 88, 264 and 880 mg/kg bw/day groups, respectively. The slightly lower values noted in the treatment groups were considered to be caused by a relatively high control value when compared to historical control data, and as these changes were not statistically significant, they were not considered related to treatment with test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 880 mg/kg bw/day, food consumption before or after correction for body weight was statistically significantly lower than controls on Days 6-12 post-coitum. This was partly caused by the remarkable low individual values for three females found dead or sacrificed in extremis, especially on Days 9-12 post-coitum. These females were found death or sacrificed on Days 12-13 post-coitum (see section on mortality).
During the remainder of the treatment period, food consumption at 880 mg/kg bw/day remained in the same range as controls. The statistically significantly lower absolute food consumption at 880 mg/kg bw/day on Days 18-21 post-coitum was not considered to be toxicologically relevant, as the change was only minimal and a similar food intake was noted at 88 mg/kg bw/day.
Food consumption before or after correction for body weight was not considered to be affected at 88 and 264 mg/kg bw/day.

Food efficiency:

not examined

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related macroscopic findings were noted for the unscheduled deaths at 880 mg/kg bw/day. At necropsy, a gastro-intestinal tract (partly) distended with gas in combination with or without a gelatinous content was observed for all five early sacrifices. The spleen of two females sacrificed on Days 12 post-coitum was reduced in size, and in one of these females, the thymus was reduced as well. Isolated gray-white foci on the lungs were noted in addition for one female.
Macroscopic examination at scheduled necropsy revealed no toxicologically relevant findings. No alterations were noted for the remaining females at 1000 mg/kg and only incidental findings, i.e. alopecia and a gray-white focus on the liver were noted for single females dosed at 88 and 264 mg/kg bw/day, respectively

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):

There were no treatment-related effects observed up to and including the highest dose level of 880 mg/kg bw/day.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on pre- or post-implantation loss by treatment up to and inlcuding 880 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no treatment-related effects observed up to and including the highest dose level of 880 mg/kg bw/day.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no treatment-related effects observed up to and including the highest dose level of 880 mg/kg bw/day.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no treatment-related effects observed up to and including the highest dose level of 880 mg/kg bw/day.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One female at 1000 mg/kg bw/day was not pregnant. All other females were pregnant with viable fetuses.

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

264 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 880 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.4 and 5.2 gram for the control, 88, 264 and 880 mg/kg bw/day groups, respectively.

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): There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 880 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no effects observed up to and including the highest dose level of 880 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to and including 880 mg/kg bw/day.
Mean sex ratios (males:females) were 49:51, 50:50, 41:59 and 51:49 for the control, 88, 264 and 880 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 10.7, 10.2, 10.0 and 9.9 fetuses/litter for the control, 88, 264 and 880 mg/kg bw/day groups, respectively.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The numbers of fetuses (litters) available for morphological examination were 236 (22), 225 (22), 220 (22) and 149 (15) in controls, low, mid- and high dose groups, respectively.
The only malformations observed in groups treated with the test item were a meningoencephalocele in one high-dose fetus and a small lower jaw in a mid-dose fetus. Both cases were confirmed skeletally, whereby the latter fetus had another skull anomaly as well. As these cases occurred singly and a small or absent lower jaw was seen previously (skeletally) in historical control fetuses, they were considered chance findings.
Besides the malformations above, external malformations were observed in two control fetuses. One fetus had anasarca and appeared also to be affected viscerally (diaphragmatic hernia) and skeletally (vertebral anomaly with associated rib anomaly and bent limb bones) as well.  
The other control fetus had an omphalocele and anogenital fissure and was found to have severely malaligned sternebrae at skeletal examination.
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 880 mg/kg bw/day.
Malformations were observed in three fetuses of which the dams received test item. One high-dose fetus had bent limb bones. This malformation was also found in one mid-dose fetus and three control fetuses. The other malformed fetus was a mid-dose fetus that had a skull anomaly, besides the externally noted small lower jaw, which was already previously described. The group distribution of these two malformations did not suggest a treatment relationship as both were seen previously in historical controls.
The only other skeletal malformations were found in two control fetuses and were already mentioned together with the external findings of these fetuses.
Skeletal variations occurred at an incidence of 68.2%, 82.4%, 78.5% and 75.6% per litter in controls, low, mid- and high-dose groups, respectively. All the ones noted were not considered treatment related as they occurred in the absence of a dose-dependent relationship, infrequently 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 880 mg/kg bw/day.
Visceral malformations were noted in all groups. One high-dose fetus had situs inversus and abnormal lobation of the lung. The first finding was also found in one low-dose fetus together with an absent eye, and the latter finding was observed in one mid-dose fetus together with a right-sided aortic arch. The incidence and group distribution of the above malformations did not indicate a treatment relationship and therefore all were considered to be chance findings.
The affected control fetus was the one with diaphragmatic hernia and already described in the section on skeletal malformations.
Visceral variations were observed in 16.3%, 8.0%, 8.4% and 6.2% of fetuses per litter in controls, low, mid- and high-dose groups, respectively. They all occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

880 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at the highest tested dose level of 880 mg/kg bw/day

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Historical control data (2014 -2015)

KGA GREENS							Project 511913
Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21		Mean of Study Means
Study Date Range: 2014 - 2015
Endpoint	Total	Mean	SD	Median	Min	Max	P5	P95
No of Studies	13
Total No. of Animals in the Control Group	304
No. of Animals that Died	0
No. of Animals that were Euthanized	0
No. of Animals that Aborted or Delivered	3
Percent Pregnant		98.8	2.73	100.0	90.9	100.0	97.1	100.0
No. of Animals Examined at Laparohysterectomy	301
No. Nongravid	4
No. Gravid	297
No. with Only Resorptions	2
No. of Dams with Live Fetuses	295
Mean No. Viable Fetuses/Dam		10.7	0.71	10.6	9.1	11.6	10.3	11.2
Total No. Viable Fetuses	3194
Viable Fetuses (%/Litter)		95.2	2.63	95.9	88.9	98.4	93.6	96.8
Mean No. Postimplantation Loss/Dam		0.5	0.15	0.4	0.2	0.7	0.4	0.6
Total No. Postimplantation Losses	134
Postimplantation Loss (%/Litter)		4.8	2.63	4.1	1.6	11.1	3.2	6.4
Dead Fetuses (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Early Resorptions (%/Litter)		4.7	2.62	4.1	1.6	11.1	3.2	6.3
Late Resorptions (%/Litter)		0.0	0.11	0.0	0.0	0.4	0.0	0.1
Mean No. Implantations/Dam		11.2	0.69	11.1	9.6	12.0	10.8	11.6
Mean No. Corpora Lutea/Dam		11.9	0.71	11.7	10.9	13.2	11.5	12.3
Mean No. Preimplantation Loss/Dam		0.7	0.32	0.6	0.2	1.3	0.5	0.9
Total No. Preimplantation Losses	207
Preimplantation Loss (%/Litter)		6.2	3.43	5.8	2.0	14.5	4.2	8.3
Total No. Male Fetuses	1617
Total No. Female Fetuses	1577
% Males/Litter		50.8	2.12	50.7	46.6	53.7	49.5	52.0
% Female/Litter		49.2	2.12	49.3	46.3	53.4	48.0	50.5
Mean Fetal Body Weight (g)		5.2	0.08	5.2	5.1	5.3	5.1	5.2
Mean Male Body Weight (g)		5.4	0.10	5.4	5.2	5.5	5.3	5.4
Mean Female Body Weight (g)		5.1	0.07	5.1	5.0	5.2	5.0	5.1
Mean Male Placenta Weight (g)^1		0.46	0.01	0.47	0.44	0.47	0.4	0.5
Mean Female Placenta Weight (g)^1		0.44	0.01	0.44	0.42	0.45	0.4	0.5

¹Based on 4 datasets

KGA GREENS								Project 511913
Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21
Study Date Range: 2014 - 2015
No. of Studies	13
Total No. Fetuses/Litters Examined Externally	3194	295
Total No. Fetuses/Litters Examined Viscerally	2061	295
Total No. Fetuses/Litters Examined Skeletally	2059	295
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
MALFORMATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
Total External Malformations								1	1
Total Visceral Malformations								7	7
Total Skeletal Malformations								15	15
Total Malformations								22	22
EXTERNAL
Exencephaly	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Eye(s)- Open	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
VISCERAL
Diaphragmatic Hernia	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Eye(s)- Absent and/or Small	0.1	0.26	0.0	0.0	0.9	0.0	0.2	3	3
Hydrocephaly- External	0.0	0.12	0.0	0.0	0.5	0.0	0.1	1	1
Situs Inversus	0.2	0.34	0.0	0.0	1.0	0.0	0.4	3	3
SKELETAL
Jaw- Upper Jaw Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Jaw- Lower Jaw Absent or Small	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Limb Bone(s)- Bent	0.3	0.44	0.0	0.0	1.1	0.0	0.5	4	4
Rib Anomaly	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Skull Anomaly	0.1	0.34	0.0	0.0	1.2	0.0	0.3	2	2
Sternebra(e)- Fused	0.1	0.29	0.0	0.0	1.0	0.0	0.3	2	2
Sternebra(e) Malaligned (Severe)	0.0	0.08	0.0	0.0	0.3	0.0	0.1	1	1
Sternoschisis	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1
Vertebral Anomaly With or Without Associated Rib Anomaly	0.2	0.53	0.0	0.0	1.9	0.0	0.5	3	3
Vertebral Centra Anomaly	0.1	0.22	0.0	0.0	0.8	0.0	0.2	1	1

KGA GREENS								Project 511913
Historical Control Data Rat: Crl:WI(Han) (outbred.SPF-Quality)
Gestation Day 21
	Mean of Study Means							Summary Incidence
	(% Per Litter Basis)							(Total No. Affected)
VARIATIONS	Mean	SD	Median	Min	Max	P5	P95	Fetuses	Litters
EXTERNAL
None Observed
VISCERAL
Kidney(s)- Renal Papilla(e) Absent and/or Small	0.1	0.25	0.0	0.0	0.9	0.0	0.2	2	2
Liver- Appendix	1.2	0.56	1.3	0.3	2.3	0.9	1.6	23	21
Liver- Discolored	0.1	0.30	0.0	0.0	1.0	0.0	0.3	3	3
Liver- Small Supernumerary Lobe(s)	4.0	1.96	4.0	1.3	7.7	2.8	5.2	69	58
Spleen- Supernumerary	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Thymus- Partially Undescended Horn(s)	1.3	1.55	0.8	0.0	4.3	0.3	2.2	34	23
Thyroid- Discolored	0.1	0.36	0.0	0.0	1.3	0.0	0.3	1	1
Ureter(s)- Convoluted	1.0	2.39	0.0	0.0	8.7	0.0	2.5	43	28
Ureter(s)- Dilated	0.9	2.33	0.0	0.0	8.5	0.0	2.3	44	19
SKELETAL
7th Cervical Rudimentary Rib(s)	1.7	1.34	1.2	0.0	4.4	0.9	2.5	30	26
7th Cervical Full Rib(s)	0.1	0.36	0.0	0.0	1.1	0.0	0.4	2	2
14th Full Rib(s)	5.7	4.65	5.2	0.0	13.1	2.9	8.5	88	64
14th Rudimentary Rib(s)	44.1	19.84	54.4	19.0	72.0	32.1	56.1	798	250
Metacarpal(s) and/or Metatarsal(s) Unossified	2.2	1.97	1.0	0.0	6.3	1.0	3.4	41	24
Pelvic Girdle- Caudal Shift	6.6	3.77	7.1	1.7	12.8	4.3	8.9	127	71
Rib(s)- Bent	10.6	7.78	10.2	0.8	22.3	5.9	15.3	162	85
Rib(s)- Short	0.0	0.06	0.0	0.0	0.2	0.0	0.0	1	1
Skull- Reduced Ossification	2.7	2.55	1.8	0.0	7.0	1.2	4.3	81	46
Skull- Supernumerary Site	0.0	0.14	0.0	0.0	0.5	0.0	0.1	1	1
Sternebra(e) #1, #2, #3 and/or #4 Unossified	0.2	0.31	0.0	0.0	0.8	0.0	0.3	3	3
Sternebra(e) #5 and/or #6 Unossified	0.9	1.33	0.0	0.0	4.1	0.1	1.7	37	23
Sternebrae- Malaligned (Slight or Moderate)	11.1	5.72	8.9	4.4	21.3	7.6	14.5	188	131
Sternum- Supernumerary Ossification Site	0.1	0.31	0.0	0.0	1.1	0.0	0.3	1	1
Vertebral Centra- Reduced Ossification	0.6	0.88	0.4	0.0	3.0	0.1	1.2	12	12

Summary of developmental effects

Dose level (mg/kg bw/day)	0	86	264	860
Pregnant/total dams	22/22	22/22	22/22	21/22
-early resorptions -late resorptions (% per litter)	6.3 0.0	7.6 0.0	7.2 0.8	1.0 1.7
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses only	0	0	0	0
Pre-implantation loss (number and percent)	19 (7.5%)	16 (6.7%)	28 (10.3%)	15 (9.9%)
Post-implantation loss (number and percent)	16 (6.3%)	15 (7.6%)	16 (7.9%)	3 (2.7%)
Body weight on day 21	331	320	323	314
Body weight gain day 6-21 (%)	48	44	46	44
Gravid uterine weight (g)	76.6	71.9	72.4	69.7
Mean live offspring (number)	10.7	10.2	10.0	9.9
Live offspring (percent)	93.7	92.4	92.1	97.3
Mean fetal/pup body weight males (g)	5.5	5.5	5.5	5.3
Mean fetal body weight females	5.2	5.1	5.3	5.0
Mean fetal body weight (sexes combined)	5.3	5.3	5.4	5.2
Malformations (including runts) number and percent of fetuses per litter - external - visceral - skeletal	4 (2.3%) 2 (0.8%)  1 (0.8%) 4 (3.2%)	1 (0.6%) 0 (0.0%) 1 (0.6%) 0 (0.0%)	3 (2.9%) 1 (0.5%) 1 (0.9%) 2 (2.4%)	3 (3.2%) 1 (0.7%) 1 (1.1%) 1 (1.3%)
Variations (% per litter) -external -soft tissue -skeletal	0 16.3 68.2	0 8.0 82.4	0 8.4 78.5	0 6.2 75.6

Analytical verification of the dose concentrations

Accuracy

The concentrations analysed in the formulations of the three dose groups were not in agreement with target concentrations (i.e. mean accuracies were not between 85% and 115%, i.e. 69%, 60% and 55% for low, mid- and high-dose groups on 11 October 2016, n = 6, 2 and 6, respectively, and 69%, 74% and 75% for low-, mid- and high-dose groups on 21 October 2016, n = 6, 2 and 6, respectively). This was possibly due to higher concentration of KGA in the test item used for calibration. Test item used for calibration was either taken from a different container or it was not sampled at the same time as the test item used for preparation of formulations. Test item is oversaturated with KGA and hence when not mixed properly excess of KGA deposits at the lower part of container. If test item is sampled from that part of container and used for calibration, analysed concentration falsely appears to be lower. This was confirmed in the two studies performed at the same testing facilities.

Homogeneity

For the formulation of the high-dose group prepared for use on 11 October 2016, the homogeneity was above the criteria (i.e. coefficient of variation was 16%) and therefore additional formulation was prepared on 21 October 2016.

The formulations of the low and and the high dose groups for use on 21 October 2016 were homogeneous (i.e. coefficient of variation≤ 10%).

Based on the KGA deposits at the lower part of the container, used for calibration, as the test item was constantly mixing while being weighed in for preparation of formulations and as the formulations were homogeneous, the formulations as used for dosing of Group 2, Group 3 and Group 4 were considered to be adequate and on target.

 

Applicant's summary and conclusion

Conclusions:

In the GLP-compliant guideline study with rats, no develomental effects were observed at the highest dose level of 880 mg/kg bw/day. Maternal toxicity was observed at the highest dose level, manifested as mortality, clinical signs and reduced body weight and body weight gain. The NOAEL for developmental toxicity was set at the highest dose level of 880 mg/kg bw/day, while the NOAEL for maternal toxicity was set at 264 mg/kg bw/day.

Executive summary:

In a GLP-compliant OECD guideline 414 study, groups of pregnant Crl:WI(Han) rats were administered the test substance by oral gavage at dose levels 80, 264 and 880 mg/kg bw/day from Day 6 to Day 20 (inclusive) post-coitum. One female died and five were euthanised in extremis at the highest dose level. At necropsy, a gastrointestinal tract (partly) distended with gas, with or without a gelatinous content, was observed in all early sacrifices. The remaining females showed a number of clinical signs, which were also seen in females euthanized or found dead, such as hunched posture, rales, piloerection, quick breathing, lethargy, decreased locomotor activity, squeaking and/or chromodacryorrhoea. In addition, body weight (gain) was lower at 1000 mg/kg bw/day from Day 9 post-coitum onwards and food consumption, both absolute and relative, was reduced on Days 6-12 post-coitum. No toxicologically relevant macroscopic findings were noted. Based on this the NOAEL for maternal toxicity was set at the mid-dose of 264 mg/kg bw/day.

No developmental toxicity was observed at any dose levels. No toxicologically relevant changes in the number of pregnant females, corpora lutea and implantation sites, or pre- or post-implantation loss were noted and litter size, sex ratio and fetal body weights were considered to be unaffected. Moreover, no treatment related fetal external, visceral or skeletal malformations or variations were noted by treatment up to and including the highest dose level of 880 mg/kg. Based on this, the NOAEL for developmental toxicity was set at the highest dose level of 880 mg/kg bw/day.