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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1988-09-12 to 1989-09-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as a reliable without restriction because it was conducted according to or similar to OECD TG 414.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Animal groups did not consist of a sufficient number per group. According to OECD Guideline 414, animal groups must contain 20 dams per group.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Oily liquid
Details on test material:
Read across to Distillate Aromatic Extracts

- Name of test material (as cited in study report): 318 Isthmus Furfural extract
- Substance type: Distillate aromatic extract (DAE)
- Physical state: Liquid
- Lot/batch No.: CRU# 86187
- Expiration date of the lot/batch: 1991-04-03
- Density: 0.98 g/mL

Rabiolabelled surrogates:
[Ring-UL-14C]Carbazole (9.3 mCi/mmole)
[3H-1,3,6]Benzo(a)pyrene (69.02 Ci/mmole)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Kingston, New York)
- Age at study initiation: Approximately 9-weeks old
- Fasting period before study: No
- Housing: Individually housed; animals in Group 9 were housed individually in a metabolism cage on gestation days 10 through 12
- Diet (e.g. ad libitum): Ad libitum; Purina Certified Rodent Chow #5002
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22
- Humidity (%): 40 to 60
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Intact dorsal surface
- Type of wrap if used: No wrap was used
- Time intervals for shavings or clippings: Animals were clipped on gestation day 0 and once weekly thereafter

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No washing was performed
- Time after start of exposure: Test material was not removed after exposure.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Group 2 (8 mg/kg day): 10 µL syringe (calibrated in 0.2 µL); Group 3 (30 mg/kg/day): 25 µL (calibrated in 0.5 µL); Group 4 (125 mg/kg/day): 50 µL (calibrated in 1 µL); Group 5 (500 mg/kg/day) and 8 (125 mg/kg/day): 250 µL syringe (calibrated in 5 µL); Group 6 (1000 mg/kg/day): 500 µL syringe (calibrated in 10 µL); and Group 9 (1000 mg/kg/day plus two radiolabelled surrogates): digital 250 µL Gilson Microman Model M250 micropipetter

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, Elizabethan-style collars
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data reported.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Cohabitation period lasted until 113 presumed pregnant rats were obtained.
- Verification of same strain and source of both sexes: No
- Proof of pregnancy: Females were examined every morning during mating for vaginal sperm plugs. The drop-pans under the animals cages also were checked for expelled vaginal plugs daily. Vaginal lavage fluid was examined for the presence of spermatozoa in those dams that exhibited a vaginal plug either in situ or in the drop-pan. Females with the presence of spermatozoa and a vaginal plug were considered to be at day 0 gestation and placed in individual housing units.
Duration of treatment / exposure:
The test material was applied to dose groups 2 through 4 and group 8 on gestation days 0 through 19. Dose group 5 was administered the test material on gestation days 0 through 16. Dose group 6 was administered the test material on gestation days 10 through 12. Dose group 9 (the test material plus two radiolabelled surrogates [14Ccarbazole and 3H-benzo(a)pyrene]) was applied on gestation days 10 through 12.

Initially, administration of the test material for group 5 was scheduled for gestation days 0 through 19; however, treatment was discontinued on gestation 16 due to a high incidence for resorption.
Frequency of treatment:
Daily
Duration of test:
The acclimation period started on 30 August, 1988. The visceral examinations ended on 21 September, 1989.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 8, 30, 125, 500, 1000, or 1000 plus two radiolabelled surrogates mg/kg/day
Basis:
other: Applied dermally to skin
No. of animals per sex per dose:
Control group (0 mg/kg/day): 15 animals
Group 2 (8 mg/kg/day): 15 animals
Group 3 (30 mg/kg/day): 15 animals
Group 4 (125 mg/kg/day): 15 animals
Group 5 (500 mg/kg/day): 15 animals
Group 6 (1000 mg/kg/day): 15 animals
Group 7 (0 mg/kg/day): 10 animals
Group 8 (125 mg/kg/day): 10 animals
Group 9 (1000 mg/kg/day plus 14Ccarbazole and 3H-benzo(a)pyrene): 3 animals
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on study results from Study 61737 (Thirteen-week administration of 318 Isthmus Furfural extract to rats)
- Rationale for animal assignment (if not random): Animals in dose groups 1 through 8 were randomly assigned based on a computer generated table for a stratified sample size of 6 (dose groups 1 through 6) or 2 (dose groups 7 and 8). No randomization tables were used for dose group 9.
- Other: Groups 2 through 6 and dose group 8 were administered 3, 30, 125, 500, or 1000 mg/kg/day. The amount of test material applied was calculated using the most recent body weight. Dams were clipped on the dorsal surface, and the test material was dispensed evenly over the test site using a syringe without a needle. Animals were fitted with Elizabethan-style collars. The control groups (dose groups 1 and 7) were clipped and collared in a similar fashion. The dorsal skin of each rat was stroked with the tip of a syringe, but no test material was applied. A ninth dose group was used, in which dams were applied the base oil on gestation days 10 through 12 at a dose level of 1000 mg/kg/day plus two radiolabelled surrogates (14C-carbazole and 3H-benzo(a)pyrene).

Postnatal dams were allowed to deliver their pups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once a day throughout gestation until sacrifice
- Cage side observations included: pathosis, abortion, premature delivery, dystocia, and/or death. Dams also were observed on post partum days 0 through 4 for pathosis and/or death.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 3, 6, 10, 13, 16, and 20, except for dose group 9. Body weights for dose group 9 were recorded on gestation days 0, 3, 6, and 10.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; food consumption was measured on gestation days 0 to 3; 3 to 6; 6 to 10; 10 to 13; 13 to 16; and 16 to 20, except for dose group 9. Food consumption was not recorded for dose group 9.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 for the prenatal groups; sacrificed on postpartum day 4 or as soon as last pup in litter had died; bioavailability dams were sacrificed on gestation day 13. All dams were sacrificed by over-exposure to diethyl ether.
- Organs examined: Thoracic and abdominal cavities were exposed, and reproductive organs were grossly examined. Liver and thymus weights were recorded.

OTHER: Haematology and clinical chemistry parameters were examined in 10 dams in dose group 1 to 4, as well as the nine dams in dose group 5. Haematology samples were analyzed for haematocrit, haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, platelet count, red blood cell count, and white blood cell out. Measured clinical chemistry endpoints are presented in the table below. Blood samples were collected at the time of sacrifice from the abdominal aorta.

For the bioavailability group, blood, thymus, liver, small intestines, large intestines, kidneys, stomach, and ovaries, were collected for radioactivity measurements. Placentas, embryos, amniotic fluid, and yolk sac were pooled for each dam. Urine, cage wash, and faecal samples were analyzed for radioactivity. Dermal absorption of each of the two radiolabelled surrogates was measured by summing the total of each found in the urine, cage washes, faeces, and collected maternal and embryonic tissues at the end of 72 hours. Tissue concentrations of carbazole and benzo(a)pyrene were calculated based on the radioactivity found per gram or millilitre and the specific activity of 14C-carbazole and 3H-BaP. The total amount of radiolabelled surrogate in the tissues was calculated as a percent of the total applied radioactive dermal dose (over three days).
Ovaries and uterine content:
PRENATAL DAMS

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
- Other: Live and dead foetuses

The uterus was excised and examined for the total number of implantations for postnatal dams.
Fetal examinations:
FOETUSES FROM PRENATAL DAMS

- External examinations (anomalies, malformations, and variations): Yes: all per litter
- Soft tissue examinations (anomalies, malformations, and variations): Yes: half per litter
- Skeletal examinations (anomalies, malformations, and variations): Yes: half per litter
- Head examinations: No data

Each foetus was weighed and sexed.

FOETUSES FROM POSTNATAL DAMS
On postpartum day 0, pup were examined for external malformations and variations. Pups were observed daily for the presence of milk in their stomachs; absence of milk was recorded. Pup weight was measured on postpartum days 0 and 4. All pups were preserved at sacrifice for possible skeletal examination. Additionally, intact pups found dead during the postpartum period were preserved for skeletal evaluation.
Statistics:
An ANOVA followed by Fisher's Exact or Dunnett's test was used to evaluated maternal biophase and caesarean section data, as well as foetal body weights. Foetal skeletal and visceral data were entered into the Grosse System and statistically evaluated by ANOVA followed by group comparisons using Fisher's Exact Test. Liver and thymus weights were statistically evaluated using Tukey's test. Serum chemistry data was analyzed using the Grosse Clinpath System, and haematology data were analyzed using SAS procedures. The F-test was used to do an ANOVA on the haematology and serum chemistry data obtained from the control and treated groups, followed by a Tukey's multiple comparison test. In general, differences between the control and treated groups were considered statistically significant if the probability of difference being due to chance was less than 5%.
Historical control data:
No data reported.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The following signs of toxicity were considered treatment-related. Data are presented in the tables below.

Increased red vaginal discharge (dose-related; the total incidence of red vaginal discharge over the 20 day gestation period was 0, 1, 4, 9, 12, 4, 0, 2, for dose groups 1, 2, 3, 4, 5, 6, 7, and 8, respectively)
Decreased net body weight gain and food consumption (125 and 500 mg/kg/day)
Decreased thymus weight (prenatal groups ≥125 mg/kg/day)
Increased liver weight (prenatal groups ≥ 125 mg/kg/day)
Increased aberrant haematology and serum chemistry values (125 and 500 mg/kg/day)
Increased number of dams with resorptions (125 and 500 mg/kg/day)
Increased uterine death (≥30 mg/kg/day)

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: other:
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Basis for effect level:
other: maternal toxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Effect level:
8 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The following signs of toxicity were considered treatment-related. Data are presented in the tables below.

Decreased foetal body weight (≥125 mg/kg/day)
Increased external and skeletal anomalies (1000 mg/kg/day)

Effect levels (fetuses)

Remarks on result:
other: see Details on embyotoxic/teratogenic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

 

 

Mean (±SD) Maternal Body Weight (grams)

Gestation Day

Dose (mg/kg/day)

Group 1

0 (N=13)

Group 2

8 (N=13)

Group 3
30 (N=14)

Group 4

125 (N=13)

Group 5

500 (N=10)

Group 6

1000 (N=13)

Group 7

0 (N=13)

Group 8

125 (N=10)

Day 0

256.0±11.1

263.2±13.9

259.9±10.2

262.9±9.8

262.0±15.0

266.9±11.8

271.9±17.0

272.9±14.1

Day 3

268.7±10.9

268.3±15.7

257.7±13.6

246.7**±14.8

243.2**±19.0

281.1±13.0

283.9±29.5

252.0**±16.3

Day 6

282.8±11.9

278.9±15.7

272.2±15.1

263.1**±10.6

255.5**±16.6

297.1±14.5

303.5±21.0

265.4**±12.4

Day 10

299.5±12.4

296.1±21.1

290.1±14.6

280.4*±10.9

271.3**±15.8

314.9±16.1

323.6±20.5

274.0**±9.2

Day 13

318.8±14.2

316.5±23.0

305.8±15.3

291.7**±11.3

278.1**±20.0

315.8±19.0

343.0±24.3

285.2**±11.0

Day 16

339.5±20.7

341.0±26.3

327.8±20.6

292.1**±17.3

267.9**±31.2

335.1±19.3

372.8±20.6

292.5**±15.2

Day 20

299.8±39.8

403.9±29.6

378.5±33.4

312.5**±26.6

288.5**±29.0

372.1±29.4

426.4±38.0

304.2**±24.4

* Statistically different from control, p<0.05.

** Statistically different from control, p<0.01.

 

Mean (±SD) Maternal Body Weight Gain (grams)

Gestation Days

Dose (mg/kg/day)

Group 1

0 (N=13)

Group 2

8 (N=13)

Group 3
30 (N=14)

Group 4

125 (N=13)

Group 5

500 (N=10)

Group 6

1000 (N=13)

Group 7

0 (N=13)

Group 8

125 (N=10)

Days 0 to 3

13±6

5±8

-2**±9

-16**±15

-19**±10

14±7

12±5

-21**±10

Days 3 to 6

14±4

11±7

15±9

16±10

12±12

16±5

20±4

13±10

Days 6 to 10

17±5

17±9

18±7

17±6

16±7

18±5

20±9

9*±12

Days 10 to 13

19±5

20±7

16±4

11*±7

7**±7

1**±9

19±76

11*±6

Days 13 to 16

21±10

24±4

22±9

0**±15

-10**±20

19±7

30±10

7**±7

Days 16 to 20

60±21

63±7

51±15

20**±16

21**±13

37**±14

54±29

12**±15

Days 0 to 20

144±38

141±18

119±25

50**±30

27**±21

105**±24

154±27

31**±28

* Statistically different from control, p<0.05.

** Statistically different from control, p<0.01.

 

Mean (±SD) Maternal Uterine and Net Body Changes (grams)

Observations

Dose (mg/kg/day)

Group 1

0 (N=13)

Group 2

8 (N=13)

Group 3
30 (N=14)

Group 4

125 (N=13)

Group 5

500 (N=10)

Group 6

1000 (N=13)

Gravid uterus

74.5±27.6

77.8±7.8

61.3±26.3

14.2**±12.8

4.5**±4.3

44.1**±17.7

Carcass

325.3±18.7

326.1±25.9

317.2±11.1

298.2**±18.9

284.0**±27.3

328.0±17.2

Net weight change from day 0

69.3±13.7

62.9±15.7

57.3±7.0

35.4**±21.2

22.0**±18.3

61.0±13.8

** Statistically different from control, p<0.01.

 

Mean (±SD) Maternal Food Consumption During Gestation

Gestation Days

Dose (mg/kg/day)

Group 1

0 (N=13)

Group 2

8 (N=13)

Group 3
30 (N=14)

Group 4

125 (N=13)

Group 5

500 (N=10)

Group 6

1000 (N=13)

Group 7

0 (N=13)

Group 8

125 (N=10)

Days 0 to 3

18.0±2.1

15.4±2.2

13.1**±3.4

8.7**±5.6

9.3**±3.7

19.4±2.6

17.6±2.9

7.4**±4.2

Days 3 to 6

21.8±1.2

18.3**±2.2

17.3**±2.8

16.5**±1.9

13.0**±3.3

23.5±2.0

23.7±2.3

11.6**±2.7

Days 6 to 10

23.3±1.6

21.2±3.4

20.4**±1.9

19.4**±1.4

18.5**±1.8

24.6±2.1

25.1±2.2

15.7**±3.3

Days 10 to 13

24.7±1.7

23.8±3.4

22.5±1.8

20.9**±1.8

19.6**±2.7

18.8**±3.6

26.5±2.3

20.5**±3.2

Days 13 to 16

25.8±1.9

25.4±2.2

24.2±2.1

19.5**±5.2

15.0±**8.0

21.0*±2.8

27.8±2.3

20.5**±2.4

Days 16 to 20

27.2±2.9

28.5±2.8

27.1±2.1

23.3**±3.9

24.4±4.0

28.3±1.9

29.5±2.7

22.5**±3.4

* Statistically different from control, p<0.05.

** Statistically different from control, p<0.01.

 

Select Mean (±SD) Reproduction Data

Observations

Dose (mg/kg/day)

Group 1

0

Group 2

8

Group 3
30

Group 4

125

Group 5

500

Group 6

1000

Viable Foetuses

13.9±5.3

N=181

14.6±1.5

N=190

11.6±5.3

N=162

2.1**±2.4

N=27

0.2**±0.6

N=2

8.8**±4.2

N=114

Resorptions (N)

0.6±0.7

N=8

1.5±1.0

N=19

4.1±4.7

N=58

11.9**±4.7

N=155

15.3**±1.6

N=153

7.5**±4.8

N=98

Resorptions (%)

11.8±27.0

9.0±5.7

27.3±31.3

82.3**±19.5

98.8**±4.0

44.9**±28.7

** Statistically different from control, p<0.01.

There was a decrease in the number of viable foetuses; consequently, there is a decrease in litter size.

 

Select Mean (±SD) Organ Weights – Absolute (g)

Observations

Dose (mg/kg/day)

Group 1

0 (N=13)

Group 2

8 (N=13)

Group 3
30 (N=14)

Group 4

125 (N=13)

Group 5

500 (N=10)

Group 6

1000 (N=13)

Group 7

0 (N=10)

Group 8

125 (N=7)

Final Body Weight

325±19

326±26

317±11

298*±19

284**±27

328±17

345±23

299±13

Liver

4.6762±0.4529

4.8992±0.3399

5.1284±0.2630

5.2005*±0.4073

5.9139**±0.5091

5.3999**±0.2882

4.2801±0.4116

4.2929±0.4279

Thymus

0.0760±0.0172

0.0773±0.0206

0.0646±0.0180

0.0475**±0.0073

0.02484**±0.0054

0.0345**±0.0069

0.0585±0.0152

0.0611±0.0210

* Statistically different from control, p<0.05.

** Statistically different from control, p<0.01.

 

Select Mean (±SD) Organ Weights – Relative (%)

Observations

Dose (mg/kg/day)

Group 1

0 (N=13)

Group 2

8 (N=13)

Group 3
30 (N=14)

Group 4

125 (N=13)

Group 5

500 (N=10)

Group 6

1000 (N=13)

Group 7

0 (N=10)

Group 8

125 (N=7)

Final Body Weight

325±19

326±26

317±11

298*±19

284**±27

328±17

345±23

299±13

Liver

15.239±1.877

15.982±1.689

16.280±1.198

15.557±1.951

16.798±2.258

17.741*±1.730

14.800±1.816

12.881±1.710

Thymus

0.246±0.050

0.255±0.001

0.205±0.059

0.142**±0.026

0.081**±0.019

0.114**±0.025

0.204±0.062

0.185±0.074

* Statistically different from control, p<0.05.

** Statistically different from control, p<0.01.

 

Select Mean (±SD) Haematology Data

Observations

Group 1

0mg/kg/day

Group 2

8mg/kg/day

Group 3
30
mg/kg/day

Group 4

125mg/kg/day

Group 5

500mg/kg/day

Platelet count (x 103/µL)

1094±134

1126±72

1058±134

896±371

754*±200

-31%

White blood cells (x 103/µL)

6.5±1.3

5.7±1.6

7.2±2.2

10.2*±2.8

+57%

10.0*±3.3

+54%

* Statistically different from control; observed percent difference is shown below.

 

Select Mean (±SD) Clinical Chemistry Data

Observations

Dose (mg/kg/day)

Group 1

0 (N=10 or 9)

Group 2

8 (N=10)

Group 3
30 (N=10)

Group 4

125 (N=10)

Group 5

500 (N=8 or 9)

Urea nitrogen (mg/dL)

18.2±2.5

20.5±4.7

19.4±4.6

25.0*±3.7

38%

25.1*±5.6

38%

ALK (IU/L)

101±38

79±30

95±38

127±57

226**±72

Cholesterol (mg/dL)

91.2±15.1

94.8±12.7

92.9±13.8

111.5±21.7

127.7**±30.0

40%

Triglyceride (g/dL)

5.3±0.6

5.2±0.5

5.6±0.7

6.5**±0.6

23%

6.5**±0.6

22%

Albumin (g/dL)

3.0±0.4

3.0±0.2

3.3±0.6

4.0**±0.3

34%

4.1**±0.4
38%

Albumin/globulin ratio

1.3±0.2

1.4±0.2

1.5±0.3

1.7±0.3

1.8**±0.3

36%

Uric acid (mg/dL)

2.1±0.7

2.4±0.5

1.0±0.5

1.2*±0.4

-41%

1.4±0.4

Sodium (mmol/L)

137.1±1.9

137.2±1.0

137.8±1.1

139.3±2.7

141.1**±3.3

3%

Potassium (mmol/L)

4.17±0.31

4.47±0.16

4.51±0.30

4.63*±0.32

11%

4.83**±0.50

16%

Phosphorus (mg/dL)

5.3±0.6

6.0±0.6

5.8±0.9

7.0**±0.7

32%

6.9**±1.0

31%

Calcium (mg/dL)

9.5±0.7

9.5±0.8

9.8±0.8

10.5*±0.4

10%

10.9**±0.4

14%

Iron (µg/dL)

126±91

81±51

115±91

345**±160

174%

367**±104

192%

* Statistically different from control, p<0.05.

** Statistically different from control, p<0.01.

The selected clinical chemistry endpoints listed above fell outside of historical controls except for uric acid, sodium, potassium, and phosphorus.

 

Mean (±SD) Foetal Weights (grams)

Observations

Dose (mg/kg/day)

Group 1

0 (N=12)

Group 2

8 (N=13)

Group 3
30 (N=14)

Group 4

125 (N=8/7/6)

Group 5

500 (N=1)

Group 6

1000 (N=13/12)

Of all viable foetuses

3.5±0.2

3.5±0.2

3.3±0.2

3.0**±0.4

2.9±0.0

2.7**±0.4

Of male foetuses

3.6±0.2

3.6±0.2

3.4±0.4

3.0**±0.4

3.2±0.0

2.8**±0.4

Of female foetuses

3.4±0.2

3.4±0.3

3.2±0.2

2.8**±0.4

2.5*±0.0

2.6**±0.3

* Statistically different from control, p<0.05.

** Statistically different from control, p<0.01.

 

Select Skeletal Observations

Observations

Dose (mg/kg/day)

Group 1

0

Group 2

8

Group 3
30

Group 4

125

Group 5

500

Group 6

1000

Litters evaluated

12

13

14

8

1

13

Foetuses evaluated

181

190

162

27

2

114

Total foetal gross observations

 

 

 

 

 

 

Foetal incidence (N)

0

0

0

1

0

5**

Foetal incidence (%)

0.0

0.0

0.0

3.7

0.0

4.4

Litter incidence (N)

0

0

0

1

0

4

Litter incidence (%)

0.0

0.0

0.0

13

0

31

** Statistically different from control, p<0.01.

Five 1000-mg/kg/day foetuses from 4 litters showed various external anomalies. Two foetuses were oedematous. Three foetuses exhibited shorted limbs, shortened and missing digits, shortened trunk, cleft palate, and kinked tails. None of these effects were statistically significant; however, statistically significance was reached when evaluating the anomalies as a whole.

 

Select Skeletal Observations

Observations

Dose (mg/kg/day)

Group 1

0

Group 2

8

Group 3
30

Group 4

125

Group 5

500

Group 6

1000

Litters evaluated

12

13

14

8

1

13

Foetuses evaluated

94

99

98

15

1

60

Rib malformation

Costal cartilage misshapen

 

 

 

 

 

 

Foetal incidence (N)

0

0

0

0

0

12**

Foetal incidence (%)

0.0

0.0

0.0

0.0

0.0

20

Litter incidence (N)

0

0

0

0

0

4

Litter incidence (%)

0.0

0.0

0.0

0.0

0.0

31

** Statistically different from control, p<0.01.

 

Applicant's summary and conclusion

Conclusions:
Heavy paraffinic distillate furfural extract produced maternal, reproductive and foetal toxicity. Maternal toxicity was exhibited as vaginal discharge (dose-related), body weight decrease, reduction in thymus weight and increase in liver weight (125 mg/kg/day and higher) and aberrant haematology and serum chemistry (125 and/or 500 mg/kg/day). Evidence of potential reproductive effects was shown by an increased number of dams with resorptions and intrauterine death. DAE was developmentally toxic regardless of exposure duration as indicated by increased resorptions and decreased foetal body weights. Furthermore, when exposures were increased to 1000 mg/kg/day and given only during gestation days 10 through 12, cleft palate and ossification delays were observed. Cleft palate was considered to indicate a potential teratogenic effect of DAE.
Executive summary:

Read across justification

Read-across from DAE is based on the fact that DAEs are a by-product from the extraction of aromatic components from the vacuum distillates used to produce LBO. In that sense, they can represent a fraction derived from unrefined/poorly refined LBO that potentially has a higher content of polycyclic aromatic compounds (PACs) compared to the LBO. Since systemic effects are related to the level and profile of PACs, DAE might represent a worst-case example of effects from the poorly refined LBO.

There are no developmental toxicity studies of lubricant base oils with IP 346 > 3%. However, there is a developmental toxicity study of a distillate aromatic extract (DAE) from a heavy paraffinic vacuum distillate which can be used as a worst case basis to assess the developmental toxicity of lubricant base oils with IP 346 > 3 wt%. In this study heavy paraffinic DAE (CAS No. 64742-04-7), 318 Isthmus Furfural Extract, was tested in a dermal study during gestation days 0 to 19 for developmental effects and maternal toxicity in the Sprague-Dawley rat.

 

Nine groups of pregnant rats were divided in three groups: prenatal, postnatal and bioavailability groups. These groups are further described below. Bioavailability group procedures and results are described separately.

 

Prenatal groups: the undiluted test sample was applied without occlusion to the shaved skin of pregnant rats at doses of 8, 30, and 125 mg/kg/day on gestation days 0-19 (15/group). An additional group received the same treatment at 500 mg/kg/day on gestation days 0 through 16. Initially, administration of the test sample to the 500 mg/kg/day group was also scheduled for gestation days 0 through 19, however treatment was discontinued after gestation day 16 because a high incidence of resorption was suspected (as indicated by a red vaginal discharge observed among rats in this group). Another prenatal group received the same treatment at 1000 mg/kg/day only on gestation days 10 through 12, an interval at which the developing foetus is sensitive to teratogenic insult. A group of sham treated rats served as control. Prenatal groups were sacrificed on gestation day 20.

 

The postnatal group was exposed under the same conditions as the prenatal group. Postnatal animals (10/group) were dosed at 0 or 125 mg/kg/day on gestation days 0 through 19. Postnatal groups were allowed to deliver their offspring naturally. Pups were observed on post partum day 0 for external malformations and variations and then together with their dams, sacrificed on post partum day 4.

 

End points examined in adults included clinical signs (all groups except bioavailability group) body weight (all groups), food consumption (all groups except bioavailability group), haematology and serum chemistry (only prenatal groups), liver and thymus weights (all groups except bioavailability group), and uterine and net body weights (all groups except bioavailability group). Foetal toxicity examinations included: resorption incidences, anomalous development (gross, skeletal and visceral abnormalities) and body weight.

 

Results - Prenatal Group

 

Prenatal groups were sacrificed on gestation day 20. All mothers were necropsied and grossly examined. Uterus and ovaries were excised and examined grossly. Numbers of corporea lutea per ovary of each pregnant animal were counted. Ovaries of non-pregnant animals were grossly examined and then discarded. Number and location of implantations, early and late resorptions and live and dead foetuses were recorded.

 

Maternal toxicity: Red vaginal discharge was observed in a number of pregnant animals in all DAE-exposed groups. Although authors mentioned this as being dose related, no statistics are provided.

 

In general animals exposed at 125 and 500 mg/kg consumed less food; at this dose level significant reduction in body weight gain, net body weight gain, and gravid uterine weight occurred throughout gestation. Body weight gain was also decreased at dose level of 1000 mg/kg/day. Of the haematology parameters evaluated, platelet and white blood cell counts were significantly affected in a dose related manner. Effects on 14 of 22 analyzed serum components were noted at the 125 or/and 500 mg/kg/day dose levels.

 

Thymus weights were significantly reduced and liver weights increased at doses in excess of 30 mg/kg/day. 

 

Reproductive effects: Implantation was not adversely affected by treatment. The number of dams with no viable offspring was increased at dose levels from 125 mg/kg/day. Litter size was significantly lower and resorptions were significantly increased compared to controls at 125 mg/kg/day and higher.

 

Foetal toxicity and development: At 30 mg/kg/day, although not statistically significant, a twofold increase over controls in the number of resorptions was observed, which the authors considered as of biological relevance. Treatment at 125 mg/kg/day and at higher doses resulted in decreased mean foetal body weights. A statistically significant increase in the incidence of incompletely ossified skull bones in foetuses exposed in utero to 125 mg/kg/day was observed. 

 

When the period of exposure was restricted to gestation days 10 through 12 and the dose increased to 1000 mg/kg, defects in costal cartilage development were significantly increased. Two of 114 foetuses evaluated were oedematous and had cleft palates. The cleft palate finding was considered by the authors to be biologically significant and evidence of a teratogenic effect, basis very low historical control incidences at theirs and other laboratories.

 

Results - Postnatal Group

 

In the postnatal group (10/group), three females were found to be not pregnant, five females reabsorbed their entire litters and one dam had only two pups, which she subsequently cannibalized. The postpartum analysis of the single viable litter of this group was not meaningful.

 

Results - Bioavailability Group

 

The bioavailability group included 3 females which were dosed with14C-carbazole- and3H benzo[a]-pyrene [BaP] - labelled DAE at 1000/kg/day on gestation days 10 through 12 and were treated under the same conditions as the previous two groups. Animals were housed in metabolic cages until sacrifice; urine and faeces were collected.

 

Bioavailability group animals were sacrificed on gestation day 13. Maternal tissues collected for radioactivity measurements were: blood, thymus, liver, small intestine, large intestine, kidneys, stomach and ovaries. Placentas, embryos, amniotic fluid and yolk sacs were pooled for each dam before analysis of radioactivity.

 

Bioavailability analyses revealed that dermal absorption of both radiolabelled substrates occurred.14C-carbazole was more extensively absorbed than3H-BaP over a 72-hour period (20% and 4% of the original dose respectively after three applications). After 72 hours, about 2% of14C-carbazole and3H-BaP (2.1% and 1.8% respectively) was found in the maternal tissues, primarily in blood, large and small intestines. By comparison, less than 0.01% of each surrogate was detected in the embryo, indicating that3H-benzo[a]-pyrene and14C carbazole do not selectively accumulate in the embryo under conditions of this study. 

 

The authors concluded that under study conditions heavy paraffinic distillate furfural extract produced maternal, reproductive and foetal toxicity. Maternal toxicity was exhibited as vaginal discharge (dose-related), body weight decrease, reduction in thymus weight and increase in liver weight (125 mg/kg/day and higher) and aberrant haematology and serum chemistry (125 and/or 500 mg/kg/day). Evidence of potential reproductive effects was shown by an increased number of dams with resorptions and intrauterine death. DAE was developmentally toxic regardless of exposure duration as indicated by increased resorptions and decreased foetal body weights. Furthermore, when exposures were increased to 1000 mg/kg/day and given only during gestation days 10 through 12, cleft palate and ossification delays were observed. Cleft palate was considered to indicate a potential teratogenic effect of DAE.

This study received a Klimisch score of 1 and is classified as reliable without restriction because this study is compliant with OECD Guideline 414.