1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

other information

Study period:

25 September 1996 to 17 October 1996 / 25 March 1997 to 12 May 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Rat: The study lacks control groups and a second higher dose. However, this is of no influence on the outcome of the experiment.

Data source

Reference

Reference Type:

publication

Title:

Disposition and Excretion of 14C-AHTN and 14C-HHCB After Intravenous Administration to Sprague-Dawley Rats and Domestic Pigs

Author:

Anne Marie Api, Gretchen Ritacco, I. Glenn Sipes

Year:

2013

Bibliographic source:

International Journal of Toxicology 32(4) 288-295

Materials and methods

Objective of study:

toxicokinetics

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

Rat: No control group and no (second) higher dose

Principles of method if other than guideline:

- Principle of test (pig): The excretion of 14C-HHCB and whole-blood and plasma kinetics have been studied in a male domestic pig after an intravenous dose of 14C-HHCB at a nominal dose of 0.1 mg/kg. Samples of skin and fat were also analysed for radioactivity residues at 9, 16 and 28 days after dosing.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

EC Number:

214-946-9

EC Name:

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Cas Number:

1222-05-5

Molecular formula:

C18H26O

IUPAC Name:

4,6,6,7,8,8-hexamethyl-1H,3H,4H,6H,7H,8H-indeno[5,6-c]pyran

Test material form:

liquid: viscous

Radiolabelling:

yes

Test animals

Details on test animals or test system and environmental conditions:

Rat: Sprague-Dawley (female)
TEST ANIMALS
- Source: Charles River Uk, Ltd.
- Age at study initiation: ca. 10-11 weeks
- Weight at study initiation: 213-230 g
- Strain: Sprague-Dawley (Crl: CD®BR)
- Housing: Stainless steel cages with suspended mesh floors except for excretion/balance experiments, during which the rats were housed in glass Metabowls designed to facilitate the separation of urine and faeces and the trapping of expired air.
- Individual metabolism cages: yes
- Diet (ad libitum): LAD1, Special diet services Ltd.
- Water (ad libitum): Anglian Water mains supply
- Acclimation period: 5 days before administration of the dose
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C±2°C
- Humidity (%): 40-60%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Pig: Large white hybrid (male)
TEST ANIMAL
- Source: R. Beedles, Shadymoor, Dorrington, Shropshire, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 33 kg
- Housing: Stainless steel metabolism cage
- Individual metabolism cages: yes
- Diet: Pig grower No 1 (Dodson and Horrel Ltd) 500 grams twice daily up to day 6 and 700 grams twice daily thereafter up to day 29.
- Water: ad libitum
- Acclimation period: 12 days before administration of the dose
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 5-18°C during the 12 days prior to dosing and 14-23°C during the 29 days after dosing.

Administration / exposure

Route of administration:

intravenous

Vehicle:

other: Ethanol/Emulphor EL 620/isotonic saline (1:1:7) solution

Duration and frequency of treatment / exposure:

Rat: The test substance was administrated as a single dose received via a injection in the tail vein.
Pig: The test substance was administrated as a single intravenous injection into the ear vein.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

Rat

Doses / concentrations 2

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Remarks:

Pig (actual dose 0.101 mg/kg bw)

No. of animals per sex per dose / concentration:

Rat: 52 female rats (In the preliminary experiment 4 female rats)
Pig: 1 male

Control animals:

no

Details on dosing and sampling:

Rat:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, cage washes, liver, kidneys, carcasses
- Time and frequency of sampling: In the main study rats were sacrified in groups of 4 and 5, 15 and 30 min., and 1, 2, 4, 6, 12, 24, and 48 hours and, and 7 , 14 and 28 days. Urine and faeces were
collected at 24-hour intervals from animals sacrified at 7 days. 14C-carbon dioxide was collected by passing expired air through 2 traps containing 2-ethoxyethanol/ethanolamine. Contents of the traps were collected at 24-hours intervals up to 28 hours. Cage washes were made with water at the time of sacrifice (168 hours).

Pig:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, fat, skin
- Time and frequency of sampling: Urine was collected at 0-6 and 6-24 hour intervals up to 14 days and faeces were collected at 24-hour intervals over the same period. Blood samples (ca. 2 mL) were taken from the jugular vein at pre-dose, 10, 20, 40 minutes, 1, 2, 4, 8, 12 and 24 hours and at 2, 3 , 5, 7, 14, 21 and 28 days. Fat/skin biopsies (8 mm diameter) were taken at 9 and 16 days after administration of the dose under sedation and local anaestetic.

Results and discussion

Preliminary studies:

Rat: In a preliminary experiment 4 rats were administered single intravenous doses of non-radiolabelled HHCB. No adverse effects were observed at this dose level (2 mg/kg).

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Rat: Mean concentrations of radioactivity in plasma declined from 2.57 jig equivalents/g at 5 minutes to 1.54, 1.31, 1.04, 0.564 and 0.249 jig equivalents/g at 30 minutes, 2, 6 12 and 24 hours respectively. Between 48 hours and 14 days radioactivity declined from 0.102 to 0.00199 jig equivalents/g with a half life of 2.1 days after which concentrations declined more slowly to 0.00050 jig equivalents/g at 28 days. Analysis of plasma samples for parent compound showed that 7.2 % and 1.6 % of plasma radioactivity were associated with parent HHCB at 2 and 24 hours respectively. Mean concentrations of radioactivity in whole-blood declined from 1.58 jig equivalents/g at 5 minutes to 0.914, 0.716, 0.565, 0.332, 0.148 jig equivalents/g at 30 minutes, 2, 6 12 and 24 hours respectively. Concentrations declined to 0.0108 j.i.g equivalents/g at 7 days. Between 7 and 28 days radioactivity declined to 0.00185 ji.g equivalents/g with a half life of 8.5 days. At earlier times radioactivity in blood was primarily associated with plasma. From 7 days the major proportion of blood radioactivity was associated with the cells.

Details on distribution in tissues:

Rat: Mean concentrations of radioactivity in liver declined from 8.83 jig equivalents/g at 5 minutes to 2.32 ug equivalents/g at 4 hours. Mean concentrations of radioactivity rose to 2.39 ug equivalents/g at 6 hours and then declined to 1.09 and 0.548 ug equivalents/g at 24 and 48 hours respectively. Between 7 and 28 days mean concentrations declined from 0.121 to 0.0221 ug equivalents/g. Mean concentrations of radioactivity in kidneys declined from 4.65 ug equivalents/g at 5 minutes to 2.38, 1.26, 0.8 17 and 0.227 ug equivalents/g at 30 minutes, 2, 6 and 24 hours. Concentrations declined to 0.0237 ug equivalents/g at 7 days and to 0.004 15 ug equivalents/g at 28 days with a half life of 8.6 days. Mean concentrations of radioactivity in fat rose from 1.21 ug equivalents/g at 5 minutes to 6.64 ug equivalents/g at 2 hours after which concentrations declined to 4.20 ug equivalents/g at 6 hours and then rose to a second peak of 4.75 ug equivalents/g at 12 hours. Between 24 hours and 14 days radioactivity declined from 3.66 ug equivalents/g to 0.098 9 ug equivalents/g with a half life of 2.6 days after which concentrations declined more slowly to 0.0260 ug equivalents/g at 28 days. Analysis of fat samples for parent compound showed that most of the radioactivity (57 - 77%) was associated with HHCB. Concentrations of HHCB of 3.81, 2.45 and 0.442 ug/g were found in fat from animals sacrificed at 2 hours, 1 day and 7 days respectively.

Pig:Concentrations of radioactivity in plasma and whole-blood declined from 108 and 69.9 ng equivalents/g respectively to 34.1 and 21.3 ng equivalents/g respectively during the initial distribution phase with half-lives of ca 1.1 hours. Thereafter concentrations declined at a slower rate to 2.6 and 1.8 ng equivalents/g respectively at 48 hours. After 48 hours concentrations fell to 1.0 and 0.7 ng equivalents/g respectively at 168 hours (7 days) with half-lives of 90 - 94 hours. At later times concentrations were close to or below the limits of accurate determination (0.3 - 0.5 ng equivalents/g). There was no obvious accumulation of radioactivity in the blood cells. Most of the radioactivity in whole-blood was associated with the plasma at all sampling times.

Details on excretion:

Rat: In animals from which excreta were collected no radioactivity was detected in expired air (0 - 48 hours). A mean recovery of 91.8 % dose was recovered from other excreta and tissues. A mean of 28.1 % dose was detected in urine of which 23.3 % dose was excreted during 0 - 48 hours. In faeces a mean of 61.0 % dose was excreted of which 8.9 % was excreted during 0 - 24 hours, 25.6 % during 24 - 48 hours and 17.7 % during 48 - 72 hours. Mean recoveries of 0.01, 0.25 and 2.14 % dose respectively were found in kidneys, liver and remaining carcass.

Pig: After a single intravenous dose of ‘4C-HHCB at a nominal level of 0.1 mg/kg a total of 88.1 % dose was recovered in urine and faeces during 14 days. Most of the radioactivity 73.5 % dose was detected in the urine, with 14.6 % dose in the faeces. The radioactivity was rapidly excreted, with 72.5 % dose being recovered from excreta during the first 48 hours after dosing.

Metabolite characterisation studies

Metabolites identified:

no

Any other information on results incl. tables

Rat:

Recovery of radioactivity in excreta and tissues of rats after intravenous doses of 14C-HHCB at a dose of 2 mg/kg.

Time (hours)	Sacrifice time/animal number and sex
		168 hours
	41	42	43	44	Mean	SD
Tissues
Kidney	0.01	0.01	0.01	0.01	0.01	0.01
Liver	0.24	0.3	0.26	0.19	0.25	0.05
Carcass	1.67	3.07	2.33	1.48	2.14	0.72
Urine
0-24	19.4	16.65	12.73	21.47	17.56	3.78
24-48	4.33	6.41	6.87	5.42	5.76	1.13
48-72	1.58	3.74	3.02	1.7	2.51	1.05
72-96	0.72	1.77	1.32	0.79	1.15	0.49
96-120	0.46	0.78	0.64	0.37	0.56	0.18
120-144	0.22	0.53	0.4	0.24	0.335	0.15
144-168	0.16	0.38	0.29	0.18	0.25	0.1
Total urine	26.87	30.26	25.27	30.17	28.14	2.48
Faeces
0-24	16.65	0.27	0.17	18.68	8.98	10.11
24-48	32.23	22.6	19.98	27.63	25.61	5.44
48-72	7.48	21.56	34.35	7.42	17.7	12.94
72-96	4.21	6.06	5.43	3.14	4.71	1.3
96-120	1.68	2.11	2.53	1.37	1.92	0.51
120-144	0.84	1.78	2	0.73	1.34	0.65
144-168	0.53	1.06	1	0.56	0.79	0.28
Total faeces	63.62	55.44	65.46	59.53	61.01	4.47
Cage washings	0.15	0.17	0.33	0.14	0.2	0.09
Air traps
0-24	nd	nd	nd	nd	nd	nd
24-48	nd	nd	nd	nd	nd	nd
Total recovery	92.56	89.25	93.66	91.52	91.75	1.88

Concentations of radioactivity in tissues of rats after intravenous doses of 14C-HHCB at a dose level of 2 mg/kg.

		Tissues
Time	Fat	Kidney	Liver	Plasma	Whole-blood
5 min	1.21	4.65	8.83	2.57	1.58
15 min	1.84	3.11	5.9	1.94	1.17
30 min	3.29	2.38	4.73	1.54	0.914
1 hr	5.27	1.94	4.03	1.46	0.845
2 hrs	6.64	1.26	3	1.31	0.716
4 hrs	5.55	0.914	2.32	1.06	0.584
6 hrs	4.2	0.817	2.39	1.04	0.565
12 hrs	4.75	0.503	1.99	0.564	0.332
24 hrs	3.66	0.227	1.09	0.249	0.148
2 days	2.17	0.092	0.548	0.102	0.0644
7 days	0.575	0.0237	0.121	0.011	0.00108
14 days	0.0989	0.00985	0.0407	0.00199	0.00438
28 days	0.026	0.00415	0.0221	0.0005	0.00185

Mean concentrations of HHCB in tissues

Time	Fat	Plasma
2 hrs	3.81	0.094
1 day	2.45	0.004
7 days	0.442

Pig:

Excretion of radioactivity in urine and faeces of a pig after an intravenous injection of 14C-HHCB at a level of 0.1 mg/kg.

Results are expressed as % dose.

Time (hours)	Urine	faeces
0 -6	28.66
6-24	22.29	3.17
24-48	11.5	6.78
48-72	2.88	1.67
72-96	2.51	1.01
96-120	1.82	0.54
120-144	1.14	0.38
144-168	0.68	0.15
168-192	0.64	0.34
192-216	0.56	0.23
216-240	0.04	0.02
240-264	0.24	0.1
264-288	0.22	0.08
288-312	0.17	0.09
312-336	0.19	0.04
Total	73.5	14.6

Concentrations of radioactivity in whole-blood and plasma of a pig after an intravenous injection of 14C-HHCB at a level of 0.1 mg/kg.

Results are expressed as ng equivalents/g.

Time (hours)	Whole-blood	Plasma
0.17	69.9	108
0.33	60.6	98.2
0.67	50.5	77.7
1	37.5	58.8
2	21.3	34.1
4	11.9	18.2
8	6.8	10.6
12	5.8	8.6
24	3.3	4.9
48	1.8	2.6
72	1.2	1.9
120	0.9	1.4
168	0.7	1
336	0.5	0.5
504	0.4	0.3
672	0.3	0.3

Concentrations of radioactivity in skin and fat of a pig after an intravenous injection of 14C-HHCB at a level of 0.1 mg/kg.

Results are expressed as ng equivalents/g.

Time (days after dosing)	Skin	Fat
9	3.8	30.5
16	0.8	3.1
28	0.5	0.5

Applicant's summary and conclusion

Conclusions:

Rat: Low bioaccumulation potential based on study results. After intravenous administration of the carbon- 14 radiolabelled compound, HHCB was eliminated in urine (28.1 % dose) and faeces (61.0 % dose). At the 7 day sacrifice, 0.25 %, dose remained in the liver and 2.14 % in the carcass. Measurement of HHCB in fat showed that from 57% (2 hours) to 77% (7 days) of the tissue radioactivity was associated with parent compound. In plasma the proportion was 7% or less. This indicated that HHCB was taken up and retained by fat after dosing but was rapidly metabolised on release from the fat.

Pig: Low bioaccumulation potential based on study results. After a single intravenous dose of ‘4C-HHCB at a nominal level of 0.1 mg/kg a total of 88.1 % dose was recovered in urine and faeces during 14 days. Most of the radioactivity 73.5 % dose was detected in the urine, with 14.6 % dose in the faeces. The radioactivity was rapidly excreted, with 72.5 % dose being recovered from excreta during the first 48 hours after dosing. Whole-blood and plasma levels showed an initial distribution phase, with a half-lives of ca 1.1 hours during the first 2 hours. Thereafter concentrations declined at a slower rate. The half-lives of radioactivity during 48 to168 hours were ca 90 - 94 hours. At later times concentrations were close to or below the limits of accurate determination (0.3 - 0.5 ng equivalents/g). There was no obvious accumulation of radioactivity in the blood cells. Most of the radioactivity in whole-blood was associated with the plasma at all sampling times. Skin and underlying fat concentrations were greater than those in plasma at 9 and 16 days but were below the limit of accurate determination (0.5 ng equivalents/g) at sacrifice (28 days, 672 hours). There was no marked long-term accumulation of radioactivity in skin and fat.

Executive summary:

Rat:

In the present study in compliance with GLP, groups of four female Sprague Dawley CD rats (bodyweight range 213-230 g–age 10-11 weeks) received a single intravenous administration of 2 mg/kg bw 14C-HHCB (uniformly labelled in the aromatic ring–radiochemical purity 99%) in a 0.4 mg/ml ethanol/Emulphor EL 620/isotonic saline (1:1:7) solution in the tail vein and were sacrificed at 5, 15, 30 min and 1, 2, 4, 6, 12, 24 and 48 hr and 7, 14 and 28 days. Tissues (fat, kidney, liver) were weighed and blood was collected by cardiac puncture. Urine, faeces and air were collected from the 4 animals that were sacrificed at day 7 after every 24 hrs until 168 hours (air up to 48 hrs). The recovery of radioactivity in these 4 animals represented 91.8 % of the dose administered: 89.3% in excreta plus cage washings, 2.14% in the carcass and 0.25% in the liver.

Maximum concentrations of radioactivity were observed in all tissues at 5 min (earliest time of measurement) except for the fat where the maximum was at 2 hr. Between 48 hr and 14 days, radioactivity in the plasma and fat decreased with apparent half-lives of elimination of 2.1 and 2.6 days respectively.In the fat, the majority of radioactivity (57-77%) was associated with parent HHCB.In whole blood, concentrations declined between 7 and 28 days with a half-life of 8.5 days with the majority of the radioactivity being associated with the cells while at earlier times it was primarily associated with the plasma. In the kidneys, the decline between 7 and 28 days was with a half-life of 8.6 days. The majority of the radioactivity (53% of the dose via faeces and 23% of the dose via urine) was excreted during the first 72 hr or 48 hr post-dosing for faeces and urine, respectively. Over the entire collection period (168 hr), the excretion via these routes amounted to 61% and 28.1% for faeces and urine. Exhalation of radioactivity could not be detected. 

 

Pig:

In the present GLP compliant study, one male domestic pig (Sus scrofa of Large White Hybrid strain–age 8-12 weeks, bodyweight 33 kg) received a nominal dose of 0.1 mg/kg bw (actual dose 0.101 mg/ kg bw) 14C- HHCB (uniformly labelled in the aromatic ring–radiochemical purity >99%) in ethanol/Emulphor EL 620/isotonic saline (1:1:7) solution by intravenous injection into the ear vein. Urine was collected at 0-6 hr and 6-24 hr and every 24 hr up to 14 days and faeces were collected at 24-hr intervals up to 14 days. Blood was collected at 10, 20 and 40 min, 1, 2, 4, 8, 12, 24 hr, 2, 3, 5, 7, 14, 21, and 28 days. Biopsies of skin and underlying fat tissue were taken at 9, 16 and 28 days (day of sacrifice).The recovery of radioactivity via the excreta was 88.1 % of the administered dose.The maximum concentrations of radioactivity in whole blood and plasma were observed at 10 min earliest collection). Radioactivity decreased rapidly in blood and plasma during the initial distribution phase with half-lives of ca. 1.1 hr. Thereafter concentrations declined at a slower rate. After 48 hrs up to 168 hrs the apparent half-life of elimination was about 90-94 hrs. At later times (336-672 hrs), concentrations were close to or below the limits of accurate determination. There was no obvious accumulation of radioactivity in blood cells.In fat, the maximal concentration (earliest collection) was at 9 days. After that, the fat concentration decreased slowly, and it was 3.1 ng equiv./g 16 days after injection and 0.5 ng equiv./g after 28 days. In skin, the maximal concentration (earliest collection) was at 9 days (3.8 ng equiv./g) declining to 0.8 ng eq/g at 16 days and to below the limit of accurate measurement (0.5 ng eq/g) at 28 days (Hawkins, 1997).

Excretion: In the pig intravenous study (GLP) described above, the majority of the radioactivity (63% of the dose via urine and 10% of the dose via faeces) was excreted during the first 48 hrs. Over the entire collection period (336 hrs) the excretion via these routes amounted to 74% and 14.6 % for urine and faeces. Exhalation of radioactivity was not monitored.